Selective inhibitors of tumor-initiating cells

ABSTRACT

Described herein are novel malignancy associated gene signature biomarkers, and assays and methods thereof, to classify prognosis or malignant potential of a cancer and identify cancer-initiating cells. The malignancy associated gene signature biomarkers, assays and methods described herein provide, in part, new methodologies to screen for novel drugs for treating cancers and tumors, such as, for example, triple-negative breast tumors. Using the assays and methods described herein proteasome inhibitors, histone deacetylase inhibitors, and glycolysis inhibitors, were identified as being highly effective in altering gene expression signatures specifically in malignant or cancer-initiating cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 National Phase Entry Application ofInternational Application No. PCT/US2012/027474 filed Mar. 2, 2012,which designates the U.S., and which claims benefit under 35 U.S.C.§119(e) of U.S. Provisional Patent Application Ser. No. 61/449,353 filedon Mar. 4, 2011, the contents of each of which are incorporated hereinby reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with Government support under W81XWH-09-1-0670awarded by the Department of Defense. The Government has certain rightsin the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 4, 2013, isnamed 033393-069632 SequenceListing.txt and is 776,090 bytes in size.

FIELD OF THE INVENTION

The present invention relates to novel malignancy associated responsesignatures associated with tumors, such as basal-like breast tumors, andmethods and assays of diagnosis, prognosis, and treatment thereof.

BACKGROUND

Despite basic and clinical research directed at understanding andcontrolling breast cancer, breast cancer is still a major health threatworldwide. Basal-like breast tumors (BL-BTs) or triple-negative breastcancers (TNBCs) are an especially aggressive group of tumors often foundin young Afro-American and Hispanic women, with the shortest survival ofall breast cancer subtypes, including luminal A, luminal B, HER2,normal-like, and claudin-low tumors. These tumors comprise ˜15% of humanbreast cancers, and more than 75% of breast cancers arising in womencarrying a BRCA1 mutation.

TNBCs are estrogen receptor, progesterone receptor and HER2 negative(triple-negative), and can express different combinations of CK5, CK14,CK17, CK18 and/or EGFR. These tumors retain a mixedluminal/myoepithelial (i.e., progenitor-like) phenotype, and recentstudies indicate that TNBCs derive from transformed mammary epithelialprogenitors. Concomitant genetic ablation of RB and TP53, or BRCA1alone, in the progenitor cell compartment of the mouse mammary glandinduces the development of basal-like malignant tumors. Notably,mutations in TP53 and/or RB genes occur in over 90% of human BL-BTs.

BL-BTs have distinct morphological and cytological features. They aretypically poorly differentiated lesions with high mitotic counts,central areas of necrosis, pushing borders and prominent stromallymphocytic infiltrates. Basal-like cancer cells, which can be arrangedin solid sheets or nests, are atypical and pleomorphic, and are highlyenriched for cells with the CD44+/CD24^(low/−) phenotype, which has beenassociated with tumor-initiating potential.

Despite response to front-line chemotherapy, tumors such as BL-BTs havea grim prognosis because of early relapse within the first five years ofdiagnosis. BL-BTs rapidly acquire resistance to chemotherapy and arerefractory to endocrine therapy and HER-2 inhibitors. Therefore there isa need for identifying methods to diagnose and treat refractory tumors,such as basal-like breast tumors.

SUMMARY OF THE INVENTION

The invention provides novel malignancy associated response signaturesand assays and methods thereof to classify and diagnose tumors, such asbasal-like breast tumors and other poor prognosis tumors, using a newlyidentified gene expression signature identified in basal-like breasttumor cells. Moreover, the invention provides new methods to screen fornovel drugs for treating cancers and tumors, such as basal-like breasttumors, using, for example, proteasome inhibitors, histone deacetylaseinhibitors, glycolysis inhibitors, or a combination thereof, which wereidentified as being effective in altering the gene expression signatureof malignant cells. The invention is based, at least in part, onfindings of a genome-wide siRNA lethality screen in highly malignantprogenitor-like breast epithelial cells, which recapitulate severalbasal-like features and give rise to tumors closely resembling humanprimary BL-BTs. As shown herein, we identified core survival pathwaysand a “malignancy associated response signature” that are selectivelyassociated with tumor-initiating potential that are useful forprognostic and diagnostic applications and reveal novel therapeutictargets for more effective and enhanced treatment of poor prognosistumors, such as BL-BTs.

Accordingly, in some aspects, provided herein are, malignancy associatedresponse signatures for a cancer-initiating cell consisting essentiallyof an expression pattern of a set of biomarkers set forth in SEQ ID NOs:1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93,and 151, where at least 5 of the 23 biomarkers have increased expressioncompared to a reference value.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is a breast cancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is a triple-negative breastcancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is a Luminal B breastcancer-initiating cell

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is an epithelial breastcancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the expression of the at least 5 of the 23 biomarkers isincreased at least 1.8-fold compared to the reference value.

Also provided herein, in some aspects, are malignancy associatedresponse signatures for a cancer-initiating cell comprising anexpression pattern of a set of at least 10 biomarkers set forth in SEQID NOs: 1-154, where at least 10 of the 154 markers have increasedexpression compared to a reference value.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is a breast cancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is a triple-negative breastcancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is a Luminal B breastcancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is an epithelial breastcancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the expression of the at least 5 biomarkers is increased atleast 1.8-fold compared to the reference value.

In some embodiments of these aspects and all such aspects describedherein, at least 2 of the set of at least 10 biomarkers set forth in SEQID NOs: 1-154 is selected from the group consisting of SEQ ID NOs: 1-4,6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and151.

In some aspects, provided herein are malignancy associated responsesignatures for a cancer-initiating cell susceptible to proteasomal geneinhibition comprising an expression pattern of a set of at least 3biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34,38, 60, 74, 76, and 149, such that at least 3 of the 15 biomarkers haveincreased expression compared to a reference value.

In some aspects, provided herein are malignancy associated responsesignatures for a cancer-initiating cell susceptible to mitosis geneinhibition comprising an expression pattern of a set of at least 3biomarkers set forth in SEQ ID NOs: 9, 17, 22, 23, 31, 37, 35, 52, 68,80, 101, 124, 130, and 137, where at least 3 of the 14 biomarkers haveincreased expression compared to a reference value.

In some aspects, provided herein are malignancy associated responsesignatures for a cancer-initiating cell susceptible to RNA splicing geneinhibition comprising an expression pattern of a set of at least 3biomarkers set forth in SEQ ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57,61, 123, 127, and 151, where at least 3 of the 13 biomarkers haveincreased expression compared to a reference value.

In some aspects, provided herein are malignancy associated responsesignatures for a cancer-initiating cell susceptible to moleculartransport gene inhibition comprising an expression pattern of a set ofat least 3 biomarkers set forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56,64, 91, 96, 98, 108, 121, and 147, where at least 3 of the 14 biomarkershave increased expression compared to a reference value.

In some aspects, provided herein are malignancy associated responsesignatures for a cancer-initiating cell susceptible to metabolic geneinhibition comprising an expression pattern of a set of at least 3biomarkers of SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126,138, 141, and 153, where at least 3 of the 13 biomarkers have increasedexpression compared to a reference value.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is a breast cancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is a triple-negative breastcancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is a Luminal B breastcancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the cancer-initiating cell is an epithelial breastcancer-initiating cell.

In some embodiments of these aspects and all such aspects describedherein, the expression of the at least 3 biomarkers is at least 1.8-foldincreased compared to the reference value.

In other aspects, provided herein are methods of classifying a cancer ina subject in need thereof, the method comprising: a) assaying expressionof ten or more of the 154 malignancy associated response signaturebiomarkers of SEQ ID NOs: 1-154 in a biological sample obtained from thesubject having a cancer, and b) comparing the expression of the ten ormore of the 154 malignancy associated response signature biomarkers ofSEQ ID NOs: 1-154 in the biological sample obtained from the subjecthaving a cancer with a reference value, where increased expression of1.8-fold or greater of at least ten of the biomarkers in the biologicalsample obtained from the subject relative to the reference valueindicates that the cancer is classified as having a poor prognosis orbeing a malignant cancer, and absence of increased expression of1.8-fold or greater of at least ten of the biomarkers relative to thereference value indicates that the cancer does not have poor prognosisor is not a malignant cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is a breast cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is a triple-negative breast cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is a Luminal B breast cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is an epithelial breast cancer.

In some embodiments of these methods and all such methods describedherein, if the cancer is classified as having a poor prognosis or beinga malignant cancer, the method further comprises the step ofadministering at least one proteasome inhibitor to the subject.

In some embodiments of these methods and all such methods describedherein, if the cancer is classified as having a poor prognosis or beinga malignant cancer and if at least two of the ten or more genes havingincreased expression is a proteasomal malignancy associated responsesignature biomarker of SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34,38, 60, 74, 76, and 149, the method further comprises the step ofadministering at least one proteasome inhibitor to the subject.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor specifically inhibits anyof the proteasomal malignancy associated response signature biomarkersset forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74,76, and 149.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor specifically inhibits themalignancy associated response signature biomarker set forth in SEQ IDNOs: 134 (MCL-1).

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor is an siRNA or antisenseRNA agent.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor is bortezomib.

In some embodiments of these methods and all such methods describedherein, if the cancer is classified as having a poor prognosis or beinga malignant cancer, the method further comprises the step ofadministering at least one histone deacetylase inhibitor to the subject.

In some embodiments of these methods and all such methods describedherein, the at least one histone deacetylase inhibitor is an siRNA orantisense RNA agent.

In some embodiments of these methods and all such methods describedherein, the at least one histone deacetylase inhibitor is trichostatin A(TSA) or Vorinostat.

In some embodiments of these methods and all such methods describedherein, if the cancer is classified as having a poor prognosis or beinga malignant cancer, the method further comprises the step ofadministering at least one glygolytic inhibitor to the subject. In somesuch embodiments, the at least one glycolysis inhibitor is an siRNA orantisense RNA agent.

In some embodiments of these methods and all such methods describedherein, if the cancer is classified as having a poor prognosis or beinga malignant cancer, relative survival rate, relative risk of metastasis,treatment option, or any combination thereof is determined for thesubject.

In some aspects, provided herein are methods of classifying a cancer ina subject in need thereof, the method comprising: a) assaying expressionof at least five biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14,23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151 in abiological sample obtained from the subject having a cancer, and b)comparing the expression of the five or more of the 23 malignancyassociated response signature biomarkers in the biological sampleobtained from the subject having a cancer with a reference value, whereincreased expression of 1.8-fold or greater of at least ten of thebiomarkers in the biological sample obtained from the subject relativeto the reference value indicates that the cancer is classified as havinga poor prognosis or being a malignant cancer, and absence of increasedexpression of 1.8-fold or greater of at least ten of the biomarkersrelative to the reference value indicates that the cancer does not havepoor prognosis or is not a malignant cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is a breast cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is a triple-negative breast cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is a Luminal B breast cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is an epithelial breast cancer.

In some embodiments of these methods and all such methods describedherein, if the cancer is classified as having a poor prognosis or beinga malignant cancer, the method further comprises the step ofadministering at least one proteasome inhibitor to the subject.

In some embodiments of these methods and all such methods describedherein, if the cancer is classified as having a poor prognosis or beinga malignant cancer and if at least one of the five or more genes havingincreased expression is a proteasomal malignancy associated responsesignature biomarker of SEQ ID NOs: 1, 2, 4, 7, 29, 33, 34, and 38, themethod further comprises the step of administering at least oneproteasome inhibitor to the subject.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor specifically inhibits anyof the proteasomal malignancy associated response signature biomarkersset forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74,76, and 149.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor specifically inhibits themalignancy associated response signature biomarker set forth in SEQ IDNOs: 134 (MCL-1).

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor is an siRNA or antisenseRNA agent.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor is bortezomib.

In some aspects, provided herein are methods of classifying a cancer ina subject in need thereof, the method comprising: a) assaying expressionof at least three malignancy associated response signature biomarkersset forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74,76, and 149 in a biological sample obtained from the subject having acancer, and b) comparing the expression of the at least three malignancyassociated response signature biomarkers in the biological sampleobtained from the subject having a cancer with a reference value, whereincreased expression of 1.8-fold or greater of at least three of thebiomarkers in the biological sample obtained from the subject relativeto the reference value indicates that the cancer is classified as havinga poor prognosis or being a malignant cancer, and absence of increasedexpression of 1.8-fold or greater of at least three of the biomarkersrelative to the reference value indicates that the cancer does not havepoor prognosis or is not a malignant cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is a breast cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is a triple-negative breast cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is a Luminal B breast cancer.

In some embodiments of these methods and all such methods describedherein, the cancer is an epithelial breast cancer.

In some embodiments of these methods and all such methods describedherein, if the cancer is classified as having a poor prognosis or beinga malignant cancer, the method further comprises the step ofadministering at least one proteasome inhibitor to the subject.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor specifically inhibits anyof the proteasomal malignancy associated response signature biomarkersset forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74,76, and 149.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor specifically inhibits themalignancy associated response signature biomarker set forth in SEQ IDNOs: 134 (MCL-1).

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor is an siRNA or antisenseRNA agent.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor is bortezomib.

Also provided herein in some aspects, are assays comprising the stepsof: a) measuring expression of ten or more of the 154 malignancyassociated response signature biomarkers set forth in SEQ ID NOs: 1-154in a biological sample obtained from a subject having cancer, and b)comparing the expression of the ten or more of the 154 malignancyassociated response signature biomarkers set forth in SEQ ID NOs: 1-154in the biological sample obtained from the subject having a cancer witha reference value, where increased expression of at least ten of themeasured biomarkers in the biological sample obtained from the subjectrelative to the reference value diagnoses the patient as having a poorprognosis or malignant cancer, and absence of increased expression of atleast ten of the measured biomarkers relative to the reference valuediagnoses the patient as not having a poor prognosis or malignantcancer.

In some embodiments of these assays and all such assays describedherein, the increased expression of the ten or more of the 154malignancy associated response signature biomarkers in the biologicalsample determines a prognosis for the subject having the cancer.

In some embodiments of these assays and all such assays describedherein, the prognosis comprises relative survival rate, relative risk ofmetastasis, treatment option, or any combination thereof.

In some embodiments of these assays and all such assays describedherein, the cancer is a breast cancer.

In some embodiments of these assays and all such assays describedherein, the cancer is a triple-negative breast cancer.

In some embodiments of these assays and all such assays describedherein, the cancer is a Luminal B breast cancer.

In some embodiments of these assays and all such assays describedherein, the cancer is an epithelial breast cancer.

In some embodiments, if the subject is diagnosed as having a poorprognosis or malignant cancer, the subject is administered at least oneproteasome inhibitor.

In some embodiments of these assays and all such assays describedherein, if the subject is diagnosed as having a poor prognosis ormalignant cancer and if at least two of the ten or more genes havingincreased expression is a proteasomal malignancy associated responsesignature biomarker of SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34,38, 60, 74, 76, and 149, the subject is administered at least oneproteasome inhibitor.

In some embodiments of these assays and all such assays describedherein, the at least one proteasome inhibitor specifically inhibits anyof the proteasomal malignancy associated response signature biomarkersset forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74,76, and 149.

In some embodiments of these assays and all such assays describedherein, the at least one proteasome inhibitor specifically inhibits themalignancy associated response signature biomarker set forth in SEQ IDNOs: 134 (MCL-1).

In some embodiments of these assays and all such assays describedherein, the at least one proteasome inhibitor is an siRNA or antisenseRNA agent.

In some embodiments of these assays and all such assays describedherein, the at least one proteasome inhibitor is bortezomib.

In some embodiments of these assays and all such assays describedherein, if the subject is diagnosed as having a poor prognosis ormalignant cancer, the subject is administered at least one histonedeacetylase inhibitor.

In some embodiments of these assays and all such assays describedherein, the at least one histone deacetylase inhibitor is an siRNA orantisense RNA agent.

In some embodiments of these assays and all such assays describedherein, the at least one histone deacetylase inhibitor is trichostatin A(TSA) or Vorinostat.

In some embodiments of these assays and all such assays describedherein, if the subject is diagnosed as having a poor prognosis ormalignant cancer, the subject is administered at least one glygolyticinhibitor.

In some embodiments of these assays and all such assays describedherein, the at least one glycolysis inhibitor is an siRNA or antisenseRNA agent.

Provided herein, in other aspects are assays comprising the steps of:(a) dividing a cell culture grown from a biopsy obtained from a subjecthaving cancer into at least 5 separate cultures; (b) exposing each ofthe at least 5 separate cultures to a different inhibitory agent,wherein each of the different inhibitory agents specifically inhibits adifferent malignancy associated response signature biomarker set forthin SEQ ID NOs: 1-154; (c) growing each of the at least 5 separate cellcultures of step (b) for at least 12 hours; and (d) measuring viabilityof the cells from each of the cultures of step (c), where if the totalviability of the cells in at least 60% of the cultures is decreased byat least 25%, then the biopsy obtained from the subject comprisescancer-initiating cells.

In some embodiments of these assays and all such assays describedherein, the inhibitory agents are selected from siRNA agents, antisenseRNA agents, or small molecules, that specifically inhibit any of thecancer gene signature biomarkers set forth in SEQ ID NOs: 1-154.

In some embodiments of these assays and all such assays describedherein, the inhibitory agents specifically inhibit any of the malignancyassociated response signature biomarkers set forth in SEQ ID NOs: 1-4,6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and151.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of themalignancy associated response signature biomarkers set forth in SEQ IDNOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91,93, and 151.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of theproteasomal malignancy associated response signature biomarkers setforth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76,and 149.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of themitosis cancer gene signature biomarkers set forth in SEQ ID NOs: 9, 17,22, 23, 31, 37, 35, 52, 68, 80, 101, 124, 130, and 137.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of theRNA splicing malignancy associated response signature biomarkers setforth in SEQ ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57, 61, 123, 127,and 151.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of themolecular transport malignancy associated response signature biomarkersset forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56, 64, 91, 96, 98, 108,121, and 147.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of themetabolic malignancy associated response signature biomarkers set forthin SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and153.

Also provided herein, in some aspects, are methods for treating a cancerin a subject in need thereof comprising the step of administering to asubject having a cancer classified or diagnosed as having poor prognosisor being malignant using any of the methods or assays described herein,at least one proteasome inhibitor in a pharmaceutically acceptablecarrier.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor is an siRNA or antisenseRNA agent.

In some embodiments of these methods and all such methods describedherein, the proteasome inhibitor specifically inhibits a proteasomalmalignancy associated response signature biomarkers set forth in SEQ IDNOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.

In some embodiments of these methods and all such methods describedherein, the at least one proteasome inhibitor specifically inhibits themalignancy associated response signature biomarker set forth in SEQ IDNOs: 134 (MCL-1).

In some embodiments of these methods and all such methods describedherein, the proteasome inhibitor is bortezomib.

Provided herein, in some aspects, are methods for treating a cancer in asubject in need thereof comprising the step of administering to asubject having a cancer classified or diagnosed as having poor prognosisor being malignant, using any of the methods or assays described herein,at least one histone deacetylase inhibitor in a pharmaceuticallyacceptable carrier.

In some embodiments of these methods and all such methods describedherein, the at least one histone deacetylase inhibitor is an siRNA orantisense RNA agent.

In some embodiments of these methods and all such methods describedherein, the at least one histone deacetylase inhibitor is trichostatin A(TSA) or Vorinostat.

Provided herein, in some aspects, are method for treating a cancer in asubject in need thereof comprising the step of administering to asubject having a cancer classified or diagnosed as having poor prognosisor being malignant, using any of the methods or assays described herein,at least one metabolic inhibitor in a pharmaceutically acceptablecarrier.

In some embodiments of these methods and all such methods describedherein, the at least one metabolic inhibitor is an siRNA or antisenseRNA agent.

In some embodiments of these methods and all such methods describedherein, the at least one metabolic inhibitor specifically inhibits ametabolic malignancy associated response signature biomarker set forthin SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and153.

Also provided herein, in some aspects, are systems for obtaining datafrom at least one sample from a subject having a cancer, the systemscomprising:

a determination module configured to receive the at least one samplefrom a subject having a cancer and perform an expression analysis of tenor more malignancy associated response signature biomarkers set forth inSEQ ID NOs: 1-154 on the at least one sample to generate an expressiondata output;

a storage device configured to store the expression data output from thedetermination module;

a comparison module configured to receive the expression data output ofthe sample from the subject having a cancer and perform at least oneexpression analysis on the expression data output to determine thepresence or absence of one of the following conditions and produce acomparison data output where:

-   -   (i) the sample from the subject having a cancer has increased        expression of ten or more metabolic malignancy associated        response signature biomarkers set forth in SEQ ID NOs: 1-154; or    -   (ii) the sample from the subject having a cancer does not have        increased expression of ten or more metabolic malignancy        associated response signature biomarkers set forth in SEQ ID        NOs: 1-154; and

an output or display module for displaying a content based in part onthe comparison data output from the comparison module, where the contentcomprises a signal indicative that the sample from the subject having acancer has increased expression of ten or more metabolic malignancyassociated response signature biomarkers set forth in SEQ ID NOs: 1-154,or a signal indicative that the sample from the subject having a cancerdoes not have increased expression of ten or more metabolic malignancyassociated response signature biomarkers set forth in SEQ ID NOs: 1-154.

In some embodiments of these systems and all such systems describedherein, the content displayed on the display module further comprises asignal indicative of the subject being recommended to receive aparticular treatment regimen.

In some aspects, provided herein are methods of identifying a candidatetherapeutic agent against a cancer initiating cell comprising the stepsof: a) exposing a BPLER cell culture to a test agent, where the BPLERcell culture comprises human breast primary epithelial cells (BPE)transformed with a defined set of genetic elements; b) measuringexpression of at least ten of the 154 malignancy associated responsesignature biomarkers of SEQ ID NOs: 1-154 in the culture, c) comparingthe expression of the same at least 10 biomarkers of malignancyassociated response signature biomarkers of SEQ ID NOs: 1-154 as wasmeasured in step (b) to an expression signature reference from a BPLERcell culture that has not been exposed to the test agent, wherein adecrease of expression of at least 5 of the at least 10 genes in thetest culture compared to the expression signature reference indicatesthat the test agent is a candidate therapeutic agent against a cancerinitiating cell.

Also provided herein are therapeutic agents identified by the methods ofidentifying a candidate therapeutic agent against a cancer initiatingcell, where the agent is an siRNA agent, an antisense RNA agent, anantibody or antigen-binding fragment thereof, or a small moleculecompound.

In other aspects, provided herein are pharmaceutical compoundscomprising a therapeutic agent identified using any of the screeningmethods described herein and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E demonstrate that BPLER cells are tumor-initiating epithelialprogenitor cells that give rise to triple negative breast tumors. (1A)Schematic depicting the method of Ince and Weinberg¹⁰ used to generatepairs of genetically identical breast cancer cell lines at differentstages of transformation from normal breast epithelial cells. Breastorganoids maintained in chemically defined media (WIT and MEGM) weresequentially transformed with retroviral vectors encoding hTERT, SV40early region and RAS^(V12). (1B) Both BPLER and HMLER are triplenegative by qRT-PCR. Luminal MCF7, mesenchymal MDA-MB-231 and HER2+SKBR3 cells were used as controls. (1C) CellTiterGlo assay showingsimilar proliferation rates of BPLER, HMLER and BPE cells in WIT medium.(1D) Tumor incidence in immunocompromised mice 8 weeks after injectionof the indicated numbers of BPLER, HMLER or MCF7 cells in Nu/J mice.(1E) qRT-PCR quantification of epithelial TNBC markers in BPLER andHMLER cells. MCF7 and MDA-MB-231 cells were used as controls for luminaland mesenchymal lineage, respectively. Data for each gene here and in(1B) were normalized to β-actin and then to the highest value in thecells tested. Data in (1B, 1C, 1E) represent the mean+/−SD of 3 (1B, 1E)or 6 (1C) replicates. Immunofluorescent staining showed selectiveco-expression of CK14 and CK18 in BPLER cells. Data in (FIGS. 1B-1E) arerepresentative of at least three independent experiments. Unsupervisedhierarchical clustering of mRNA expression of six BPLER tumors thatarose in immunodeficient mice and a set of 337 human breast primarytumors (UNC337 dataset¹²) was performed. BPLER tumors clustered withTNBC.

FIGS. 2A-2G demonstrate identification of genes selectively required forsurvival of BPLER cells. (2A) Distribution of R value and BPLER Z scorefor all 17,378 genes in the siRNA primary screen library. R, ratio ofviability of BPLER vs HMLER. BPLER Z score, a measure of the deviationof BPLER viability from the plate mean. A Z score outside of the rangeof −1 to +1 is significant. Genes were considered hits if R<0.75 and theBPLER Z score was <−1.5. Shading indicates the relative selectivity ofBPLER vs HMLER lethality. (2B) Validation rates in the secondary siRNAscreen for which at least one individual siRNA from the library poolscored positive. (2C) List of confirmed highly selective BPLERdependency genes or malignancy associated response signature genes. (2D)Single sample gene set enrichment analysis (GSEA) of the or malignancyassociated response signature and highly selective hit list in 295 humanbreast primary cancers in the NKI dataset. A Z-score for the expressionof the signature genes was calculated for each sample. The scores areshown as bean-plots to compare the distributions in the tumor subtypes(Basal, basal-like; Lum, luminal; NL, normal-like). Each bean consistsof a line for each sample with the overall distribution for the subtyperepresented as a gray density shape and a black line indicating themedian Z score. The basal-like and luminal B tumors as a group hadsignificantly enhanced expression of TGS genes or the highly selectivegenes. (2E-2G) Patients were divided into two groups based on theirtumor's GSEA enrichment score. The high malignancy associated responsesignature tumors were defined by enrichment scores with p-value <0.1,and the remaining tumors were classified as low. (2E) Kaplan-Meiersurvival curves for breast cancer patients in the NKI dataset, showingshorter survival of patients with higher tumor expression of allmalignancy associated response signature genes (left) or the highlyselective subset (right). (2F) Kaplan-Meier survival curve for luminal Bbreast cancer patients in the NKI dataset showing shorter survival ofpatients with higher tumor expression of all malignancy associatedresponse signature genes (left) or the highly selective subset (right).(2G) Kaplan-Meier curve depicting metastasis-free survival for breastcancer patients in the NKI dataset, showing longer metastasis-freesurvival in patients with lower tumor expression of all malignancyassociated response signature genes (left) or the highly selectivesubset (right).

FIG. 3 depicts a malignancy associated response signature functionalinteraction network. The malignancy associated response signatureinteraction network was constructed by incorporating physical andpredicted interactions, co-localization, shared pathways and sharedprotein domains, compiled from a variety of genomic and proteomicsources using GeneMANIA²⁰. Genes were shaded according to theirparticipation in the indicated processes. The network was constructedusing Cytoscape⁴⁰.

FIGS. 4A-4H demonstrate that Epithelial TNBC cells are selectivelysensitive to proteasome inhibitor drugs. (4A) Proteasome activity inBPLER and HMLER 6 hr after treatment with the indicated dose ofbortezomib, as determined by ProteaGlo assay. At this time most cellsare viable. (4B-4C) Viability of BPLER and HMLER cells, 24 hr aftertreatment with the indicated doses of bortezomib (4B) or doxorubicin(4C). (4D) Propidium iodide DNA staining showing accumulation oftetraploid cells (BPE and HMLER) or subdiploid cells (BPLER) 24 hoursafter treatment with bortezomib (12.5 nM). (4E) Immunoblot of lysatestreated with bortezomib at the indicated dose for 24 hr. Each sample wasassessed in duplicate independent samples. (4F) Viability of normalbreast epithelial cells (BPE), ER+ (MCF7), HER2+ (BT-474), mesenchymal(MDA-MB-231, MDA-MB-436) or epithelial TNBC (4T1-E, BPLER, HCC-1143,HCC-1937) cells treated for 24 hr with bortezomib (12.5 nM) relative tovehicle control, as assessed by CellTiterGlo. (4G) Colony assay ofHCC-1143 and 4T1-E cells treated with bortezomib (12.5 nM) for 18 hoursand cultured for 2 weeks in drug-free medium. (4H) Tumorigenicity assayof 4T1-E cells treated with bortezomib (12.5 nM) or DMSO for 18 hours invitro and injected orthotopically in the mammary fat-pad of Balb/c mice.

FIGS. 5A-5H demonstrate Bortezomib targets Mcl-1 dependency in TNBCthrough induction of Noxa. (5A) Immunoblot analysis of protein lysatesfrom BPLER and HMLER treated with bortezomib (12.5 nM) or DMSO for 24hours in the presence of ZVAD-fmk (20 uM). (5B) Representative DilC1(5)staining of BPLER and HMLER treated with bortezomib (12.5 nM) for 18hours, as determined by flow cytometry. CCCP was used as positivecontrol. The graph (right) shows median intensity value of DilC1(5)staining +/−SD from 3 independent experiments. (5C) Cell viability ofBPLER cells 48 hours after transfection with siRNAs (50 nM) against theindicated genes and treated with bortezomib (12.5 nM) or DMSO for 24hours. Data for each siRNA are normalized to 1. (5D) Baseline levels ofNoxa mRNA, relative to ACTB, by qRT-PCR in normal breast epithelialcells at different stages of transformation (BPE, BPLE, BPLER (FIG. 1A))and in breast cancer cell lines of indicated subtype. (5E) Immunoblot ofprotein lysates from MCF7 (luminal) and HCC-1143 or HCC-1937 (TNBC)breast cancer cells treated with bortezomib (12.5 nM) for 24 hr. (5F)Immunoblot of protein lysates from BPLER or HMLER treated withbortezomib (12.5 nM) or DMSO for 24 hours in the presence of ZVAD andimmunoprecipitated with antibodies against Mcl-1 or Bcl-XL or rabbitIgG, showing similar and specific Mcl1/Noxa binding in BPLER and HMLER.(5G-5H) Cell viability of human breast cancer cell lines of differentsubtype 48 hours after transfection with siRNAs (50 nM) against theindicated genes or non-targeting siRNA (control). Data in (5B, 5C, 5D,5G, 5H) represent the mean+/−SD of 3 replicates. All data arerepresentative of at least 3 independent experiments.

FIGS. 6A-6J demonstrate proteasome inhibition suppresses TNBC growth invivo. (6A) Proteasome activity in protein lysates from BPLER tumors 18hours after intratumoral (i.t.), intraperithoneal (i.p) or intravenous(i.v.) treatment with bortezomib at the indicated dose, as determined byProteasomeGlo assay. Proteasome activity in BPLER treated withbortezomib in vitro is shown as control. (6B-6E) Mean tumor volume (6B,6D) in BPLER tumor-bearing mice after treatment with bortezomib or DMSOat the indicated dose, schedule and route of administration. Mediantumor weight was measured at time of sacrifice (6C, 6E).Immunohistochemistry of BPLER tumors treated intratumorally with 0.8mg/kg bortezomib or DMSO q3d, showed caspase-3 cleavage in residualepithelial cells in bortezomib treated tumors. (6F) Immunoblot analysisof protein lysates from BPLER tumors 18 hours after 1 intravenous doseof bortezomib at 1.6 mg/kg bortezomib or DMSO, showing efficient Noxainduction and caspase-3 and PARP1 cleavage after bortezomib treatment.(6G) Mean tumor volume in MB-468 tumor-bearing mice after intravenoustreatment with 1.6 mg/kg bortezomib weekly. (6H) Schematic diagramillustrating generation of mouse TNBC primary tumor fragments forbortezomib-based therapy studies. Histological analysis of V22 mousetumors sections showed typical features of human TNBC, includingepithelial differentiation, scant stroma and CK-14 positive staining.4T1-E tumors are shown as control. (6I, 6J) Mean V22 tumor volume (6I)after weekly intravenous infusion of bortezomib (1.6 mg/kg) or DMSO.Treatment was started 2 days after implantation of primary tumorfragments. Box-and-whisker plot (6J) shows median tumor weight at timeof sacrifice. The top and the bottom of each box represent the 75^(th)and 25^(th) percentile of tumor weight, respectively. Upper and lowerwhiskers represent maximum and minimum tumor weight, respectively. Theblack horizontal band in each box corresponds to median tumor weight.

FIGS. 7A-7F demonstrate HDAC inhibitors target BPLER selectively. (7A)Connectivity map (CMAP) barview of changes in expression of malignancyassociated response signature genes following in vitro treatment ofcancer cell lines. Each black line represents an independent experimentthat measured gene expression changes following treatment of anindividual cancer cell line with the indicated drugs. Shaded areas aresignificantly enriched or depleted in malignancy associated responsesignature gene expression after drug exposure (p-values in Table 5).(7B) Viability of BPLER and HMLER cells treated with Trichostatin A,Vorinostat, Loperamide, Triamterene, Resveratrol or4-5-dianilinophthalimide for 24 hr at the indicated dose. Data representthe mean+/−SD of 6 replicates. All data are representative of at leastthree independent experiments. (7C, 7D) mRNA expression, analyzed byqRT-PCR relative to ACTB, of malignancy associated response signaturegenes, predicted to be negatively regulated by TSA, in BPLER (7C) andHMLER (7D) cells 24 hr after treatment with 1 μM TSA. (7E-7F) Percentageof apoptotic cells (7E) and immunoblot analysis of PARP1 cleavage (7F)from BPLER 24 hours after treatment with DMSO, bortezomib (12.5 nM),vorinostat (10 uM) or bortezomib/vorinostat combination.

FIGS. 8A-8C demonstrate BPLER express epithelial breast tumor-initiatingcell markers, retain an epithelial phenotype and form tumors thatresemble TNBC in vivo (8A, 8B) Most BPLER and HMLER cells areCD44+/CD24^(low) (8A) and ESA+ (8B) as assessed by flow cytometry.Luminal MCF7 and mesenchymal MDA-MB-231 were used as controls. (8C)Vimentin mRNA, assessed by qRT-PCR relative to β-actin, is poorlyexpressed, but clearly present, in BPLER compared to MDA-MB-231 andHMLER cells. MCF7 cells have no detectable vimentin mRNA. Data representthe mean+/−SD of 3 replicates. Data are representative of at least threeindependent experiments. Unsupervised hierarchical clustering of mRNAexpression profiles of 6 BPLER tumors grown in immunodeficient mice, 40human breast primary tumors (classified as either basal-like ornon-basal-like), and 7 normal breast tissues (non-cancer, Richardsondataset¹³) was performed. BPLER tumors cluster with basal-like tumors,confirming data obtained with another tumor data set. Representativeimmunohistochemical staining of a BPLER tumor was performed. Intensestaining indicated positivity for the indicated antigen.

FIGS. 9A-9G depict siRNA screen optimization. (9A) Reverse-transfectionefficiency of BPLER and HMLER cells in WIT medium in 384-well standardtissue culture plates was assessed by flow cytometry using 12commercially available liposomal formulations complexed with Cy3-labeledsiRNAs. Four transfection reagents were able to introduce Cy3-labeledsiRNAs into both cell lines with ˜90% efficiency. (9B) Only Dharmafect#1 (Dharmacon) and Lipofectamine 2000 (Invitrogen) had acceptabletoxicity (<25% decrease in cell viability) when delivering controlnon-targeting siRNAs and induced cytotoxicity after transfection with acytotoxic siRNA targeting PLK1. Titration of Dharmafect #1 reducedtoxicity in both cell lines to <15% without loss of transfectionefficiency (data not shown). Thus Dharmafect #1 was chosen for thescreen and used in all subsequent experiments. (9C) Using Dharmafect #1,13 pools of siRNAs from the Dharmacon library were transfected in bothcell lines to identify additional positive controls besides PLK1.Transfection of PLK1 and KIF11 siRNA pools similarly reduced viability,measured by CellTiterGlo assay, of both BPLER and HMLER after 72 hr,whereas the other siRNAs had little effect. (9D) Optimal time forassessing cell viability was evaluated by assessing viability atindicated times after transfection. 72 and 96 hr gave similar results,preserving viability of cells treated with non-targeting siRNAs andinducing maximal cytotoxicity with PLK1 and KIF11 siRNAs. (9E)Durability of gene knockdown in BPLER/HMLER cells, transfected with 3different non-cytotoxic siRNAs and assessed by qRT-PCR analysis of mRNAexpression, showed potent silencing in both cell lines after 48 and 72hr, which was reduced after 96 hr. Based on data in (9D, 9E), 72 hr waschosen as the optimal time to perform the viability assay for thescreen. Values in (9A-9E) represent the mean+/−SD of three replicates.(9F) To determine an optimal cell density for the screen, a CellTiterGloviability assay of BPLER (upper) and HMLER (lower) cells plated for 24hr at the indicated cell numbers in 384-well plates. The CellTiterGlosignal was linear in the range from 0 to 3000 cells/well. Cells wereplated at 1000 cells/well for all screening experiments (primary andsecondary screens). All data in (9A-9F) are representative of at leastthree independent experiments. To assess reproducibility of the highthroughput screening conditions, a prescreen was performed bytransfecting cells with a custom-made siRNA library containingnon-targeting siRNAs (192 wells/plate) or si-PLK1 (192 wells/plate). TheZ′ factor was calculated⁴⁴. For both cell lines, Z′>0.7 in each of 6microplates transfected in two separate experiments. An additionalpre-screen was performed using a 500-siRNA library (Mitchison library),which returned 4 significant positive hits (data not shown). No edgeeffect was detected in any of these experiments. (9G) Ranked BPLER Zscore values for all 17,378 genes represented in the siRNA primaryscreen library. A Z score with |Z|>1 is significant. We required hits toscore with Z≦−1.5.

FIG. 10 demonstrates that enrichment of malignancy associated responsesignature genes in primary breast tumors predicts shortermetastasis-free survival. Kaplan-Meier curves depicting metastasis-freesurvival for breast cancer patients in the EMC286 dataset¹⁵⁻¹⁶, showingshorter metastasis-free survival in patients with higher tumorexpression of all malignancy associated response signature genes (left)or the highly selective subset (right). Patients from the EMC286 datasetwere divided into two groups based on their tumor's GSEA enrichmentscore. High TGS tumors were defined by enrichment scores with p-value<0.1, and the remaining tumors were classified as low.

FIGS. 11A-11C demonstrate BPLER cells are selectively sensitive toproteasome inhibitor drugs. (11A) Immunoblot analysis of protein lysatesfrom BPE, HMLER and BPLER 24 hours after treatment with bortezomib (12.5nM) or DMSO showing similar induction of proteasome-regulated genes.(11B) Immunoblot of lysates from BPE cells treated with bortezomib (12.5nM) for 24 hr. BPLER were used as control. (11C) Flow cytometry analysisof Annexin V/Propidium Iodide (PI) staining of BPE, BPLER or HMLERtreated with bortezomib (12.5 nM) and/or zVAD-fmk (20 μM) for 24 hr. Thepercentage of double-positive cells is indicated.

FIGS. 12A-12C demonstrate bortezomib induces Noxa protein expression inTNBC cells. (12A, 12B) Noxa mRNA (12A) and protein (12B) levels assessedby qRT-PCR and immunoblot, respectively, in BPLER and HMLER treated withbortezomib (12.5 nM) for 24 hr. Values in (12A) represent mean+/−SD of 3replicates. (12C) Noxa protein levels in human breast cancer cell linesof different subtype after treatment with bortezomib (12.5 nM) or DMSOfor 24 hours. All data are representative of at least three independentexperiments.

FIG. 13 demonstrates bortezomib suppresses TNBC growth in vivo. Meantumor volume in MB-468 tumor-bearing mice upon intratumoral infusion ofbortezomib (0.8 mg/kg) or DMSO q3d.

FIG. 14 depicts additional candidate drugs targeting BPLER. Relativeviability, measured by CellTiterGlo, of BPLER and HMLER cells treatedfor 24 hr with Deferoxamine, Nabumetone, L-cavananine and Hesperetin.Viability is relative to cells grown in the absence of drug. These drugswere not active. Data represent the mean and standard deviation of sixreplicates. All data are representative of at least three independentexperiments.

DETAILED DESCRIPTION

Provided herein are novel malignancy associated gene signaturebiomarkers, and assays and methods thereof, to classify prognosis ormalignant potential of a cancer and identify cancer-initiating cells.Moreover, the gene signature biomarkers, assays and methods describedherein provide, in part, new methodologies to screen for novel drugs fortreating cancers and tumors, such as, for example, triple-negativebreast tumors. Using the assays and methods described herein proteasomeinhibitors, histone deacetylase inhibitors, glycolysis inhibitors, orany combination thereof, were identified as being highly effective inaltering gene expression signatures specifically in malignant orcancer-initiating cells.

The invention is based, at least in part, on results from a genome-widesiRNA lethality screen using highly malignant progenitor-like humanbreast epithelial cells, which recapitulate several basal-like featuresand give rise to tumors closely resembling human primary triple-negativebreast cancer cells, or TNBCs, such as basal-like breast tumors. Asdemonstrated herein, core survival pathways and a “malignancy associatedresponse gene signature” selectively associated with cancer-initiatingpotential were identified that are useful for prognostic and diagnosticapplications and reveal novel therapeutic targets for more effective andenhanced treatment of poor prognosis tumors, such as triple negativebreast cancers.

Triple-negative breast cancers (TNBCs), such as basal-like breast tumors(BL-BTs), are an especially aggressive group of tumors with the shortestsurvival of all breast cancer subtypes. These tumors are enriched, inpart, in breast tumor-initiating cells (BT-ICs), rapidly acquireresistance to chemotherapy, and are refractory to endocrine therapy andHER-2 inhibitors. No biologic therapy is currently available for thesetumors.

As demonstrated herein, we discovered that a defined population of humanprogenitor-like breast primary epithelial cells acquire cancerinitiating properties and give rise to triple-negative, basal-likebreast tumors upon transformation with defined genetic elements, i.e.,expression of certain specific genes. As shown herein, the induction ofa cancer-initiating cell phenotype, such as that of a basal-like breasttumor initiating cell, depends on the cell of origin, becausetransformation of syngeneic myoepithelial-like cells with the same setof genetic elements does not induce such phenotypes.

We performed a genome-wide siRNA lethality screen to identify factors orbiomarkers on which these two cell types selectively depend forsurvival. As demonstrated herein, these studies identified a “malignancyassociated gene signature” (MARS), or “triple negative breast cancergene signature” (TGS), comprising an expression pattern of 10-154biomarkers for cancer initiating cells, such as triple negative breastcancer initiating cells. We further determined that most of the genesrequired for survival of triple negative breast cancer initiating cellsare typically up regulated in poor prognosis human primary breasttumors, including basal-like tumors and some Luminal B tumors, thusproviding novel diagnostic and prognostic signatures, assays andmethods, and therapeutic targets, as described herein. Corecancer-initiating cell specific survival pathways and biomarkersidentified herein include, but are not limited to, theproteasome-ubiquitin system, metabolic genes, histone deacetylases, RNAsplicing, and the glycolytic system, the transient inhibition of which,as shown herein, impacts triple negative breast cancer initiating cells,but did not impact normal breast epithelial cells or transformedluminal, myoepithelial or mesenchymal cells. Accordingly, also providedherein are malignancy associated response gene signatures, and assaysand methods thereof, comprising subsets of biomarker genes belonging toeach of these core survival pathways.

It was also determined that triple negative breast cancer initiatingcells are selectively and specifically sensitive to proteasome inhibitordrugs, such as bortezomib. Proteasome inhibitor drugs were demonstratedto interfere with mitosis exit and induce Noxa-dependent apoptosis.Further, the results described herein demonstrate that the selectiveresponse of cancer initiating cells to proteasome inhibition isdependent on Noxa-mediated inactivation of MCL1 (SEQ ID NO: 134), a genebelong to the malignancy associated response gene signature of 154biomarkers. As shown herein, transformed mammary epithelial cells aswell as primary human epithelial breast cancer cells selectively dependon MCL1 or MCL-1, which is antagonized by Noxa, for survival and thatproteasome inhibitor target Mcl-1 dependency in these cells. Silencingof MCL1 expression in breast cancer cell lines led to similar lethalityof specific breast cancers subtypes as was shown herein with bortezomib.Thus, MCL-1 was identified as a novel target for cancer initiating cellsthat recapitulates the effects of proteasome inhibition. Histonedeacetylase inhibitors were also found to be selectively cytotoxic fortriple-negative breast cancers. As proteasome and histone deacetylaseinhibitors are already in the clinic, the findings described herein canbe further developed for use in new indications and targeted diagnosisof and therapeutic treatments for poor prognosis cancers, such as triplenegative breast cancers, in clinical settings.

Malignancy Associated Response Signatures and Assays and Methods Thereof

Provided herein are malignancy associated response signature foridentification of cancer initiating cells and malignancy potential andcancer prognosis. These malignancy associated response signaturesprovide novel and unique combinations of biomarkers specificallyrequired for the survival of cancer-initiating cells, such astriple-negative breast cancer initiating cells. The signatures alsoprovide novel assays and methods for classifying cancers and determiningcancer prognosis.

As used herein, a “malignancy-associated response signature” (“MARS”)(also referred to herein as a “triple negative breast cancer genesignature” (TGS)) refers to a functional module of increased geneexpression associated with cancer-initiating potential comprising atleast two or more of the biomarkers provided in Table 3 (SEQ ID NOs:1-154), termed herein as “malignancy associated response signaturegenes” or “malignancy associated response biomarkers.” As describedherein, a cancer or tumor, such as a breast cancer, can be classified asMARS (+) or MARS(−), or classified as comprising cancer initiatingcells, having poor prognosis or as being malignant, by assessing theexpression levels of at least two or more, preferably at least 10, ofthe 154 malignancy associated response signature genes or biomarkersprovided herein in Table 3 (SEQ ID NOs: 1-154) in any combination, or incombination with any other prognostic or diagnostic biomarker known toone of skill in the art. Preferably, expression levels of at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 23, at least 25, at least 30, at least 35, at least40, at least 45, at least 50, at least 100, at least 125, at least 130,at least 135, at least 140, at least 145, at least 150, or more, or allof the 154 malignancy associated response signature genes describedherein are assessed. In some embodiments of all the aspects describedherein, 5-20, 10-15, 10-20, 10-25 malignancy associated signature genesare used to analyze the unknown cancer cell for its prognosis. If atleast 60% of the signature genes are expressed more than a controlnon-poor prognosis cell, i.e., the expression is increased, the tumor orcancer from which the cell or sample originated is considered to havepoor prognosis. It is also well within the ability of one skilled in theart, using the teachings provided herein, to identify additional geneshaving prognostic or predictive value for use with signatures and assaysand methods thereof described herein.

It is envisioned that any of the genes listed in Table 3 (SEQ ID NOs:1-154, SEQ ID NOs: 181-184) can be used in the assays and methodsdescribed herein, either alone or in combination with any othermalignancy associated response signature biomarker or other prognosticmarker. Although information concerning the expression of as few as onebiomarker is expected to provide useful information, confidence in theaccuracy of the classification of a cancer or tumor as MARS(+) orMARS(−) increases when more biomarkers are included. Preferably at least10 or more of the 154 TGS biomarkers listed in Table 3 (SEQ ID NOs:1-154, SEQ ID NOs: 181-184) are used in prognostic and diagnosticanalyses of a cancer sample as described herein. Cancers can be analyzedwith respect to the expression of specific groups or subsets, of MARSbiomarkers, including from SEQ ID NO: 1 to SEQ ID NO: 154 of the geneslisted in Table 3, in any combination.

For example, in some aspects and embodiments, a malignancy associatedresponse signature can comprise only or consist essentially of genes orbiomarkers involved in a specific pathway, such as the exemplarysurvival pathways depicted in FIG. 3, e.g., the proteasomal pathway. Insome aspects and embodiments, a malignancy associated response signaturecan comprise or consist essentially of the 23 biomarkers of Table 2A(SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78,84, 91, 93, and 151) described herein as having high specificity or highselectivity for cancer-initiating potential or cells. It is well withinthe ability of one of skill in the art to select specific combinationsof MARS genes for analysis from among the genes listed in Table 3 (SEQID NOs: 1-154). In the interest of brevity, every possible combinationof genes suitable for use in the methods described herein are notexpressly listed. Nevertheless, it should be understood that every suchcombination is contemplated and is within the scope of the methodsdescribed herein. It is specifically envisioned that any combination ofMARS biomarkers found to be differentially expressed in poor prognosiscancers, for example, triple negative breast cancer samples, areparticularly useful for analysis and in the methods described herein.

Accordingly, in some aspects provided herein are malignancy associatedresponse signatures for a cancer initiating cell, such as a breastcancer initiating cell, comprising or consisting essentially of anexpression pattern or expression pattern analysis of a set of at leastten biomarkers set forth in SEQ ID NOs: 1-154, where at least 10 of the154 biomarkers have increased expression or are increased compared to areference value from a reference tissue or sample. In those embodimentsof the aspects described herein, where a malignancy associated responsesignature is said to consist essentially of a particular set ofbiomarkers, one of skill in the art will understand that additionalcontrol genes, control well, replicates, or the like biomarkers can beadded to the signature for the purposes described herein.

In some embodiments of these aspects, at least two of the set of atleast ten biomarkers set forth in SEQ ID NOs: 1-154 is selected from thegroup consisting of SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37,38, 66, 72, 77, 78, 84, 91, 93, and 151.

In some embodiments of these aspects and all such aspects describedherein, the expression of the at least ten biomarker is increased atleast 1.8 fold compared to the reference value.

The 23 biomarkers of Table 2A (SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29,33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151) were determined tohave high specificity or high selectivity for cancer-initiatingpotential or malignant potential of cells, as described herein.Accordingly, in some aspects, provided herein are malignancy associatedresponse signatures for a cancer-initiating cell comprising orconsisting essentially of an expression pattern of a set of at leastfive biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29,33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151, where at least fiveof the 23 biomarkers have increased expression compared to a referencevalue. The number of biomarkers selected for expression analysis can beat least 4, at least 5, at least 6, at least 7, at least 8, least 9, atleast 10, at least 11, at least 12, at least 13, least 14, at least 15,at least 16, at least 17, at least 18, least 19, at least 20, at least21, at least 22, or include all 23 biomarkers, in different embodiments.

In some embodiments of these aspects and all such aspects describedherein, the expression of the at least five of the 23 biomarkers isincreased at least 1.8 fold compared to the reference value.

The term “expression” refers to the cellular processes involved inproducing RNA and proteins and as appropriate, secreting proteins,including where applicable, but not limited to, for example,transcription, translation, folding, modification and processing.“Expression products” include RNA transcribed from a gene andpolypeptides obtained by translation of mRNA transcribed from a gene,and such expression can be detected using methods known to one of skillin the art. Accordingly, an “expression pattern,” as used herein, refersto a specific, defined subset of genes or biomarkers of a givenmalignancy associated response signature being expressed or notexpressed by a cell or cellular sample.

The terms “increased,” “increase” or “enhance” in connection withexpression of the malignancy associated response signature markers areall used herein to generally mean an increase by a staticallysignificant amount. For the avoidance of any doubt, the terms“increased”, “increase” or “enhance” or “activate” means an increase ofat least 10% as compared to a reference level, for example an increaseof at least about 20%, or at least about 30%, or at least about 40%, orat least about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90% or up to and including a 100%increase or any increase between 10-100% as compared to a referencevalue or level, or at least about a 1.5-fold, at least about a 1.6-fold,at least about a 1.7-fold, at least about a 1.8-fold, at least about a1.9-fold, at least about a 2-fold, at least about a 3-fold, or at leastabout a 4-fold, or at least about a 5-fold, at least about a 10-foldincrease, any increase between 2-fold and 10-fold, at least about a25-fold increase, or greater as compared to a reference level. In someembodiments, an increase is at least about 1.8-fold increase over areference value.

As is understood by one of ordinary skill in the art, the degree towhich increased expression can be detected is dependent on themethodology used for measuring expression of a signature biomarker.Microarray platforms, for example, which can comprise many thousands ofprobes corresponding to a particular biomarker, can detect even subtlechanges in gene expression, such that a 1.5-fold or greater change inexpression is deemed significant, as described herein. PCR basedplatforms, including real-time quantitative analyses, are also highlysensitive to such small changes in gene expression. Other methods ofdetecting or measuring changes in gene expression, such as, for example,Northern blot analyses are not as sensitive, such that small increasesare generally not thought to be reliable or valid. Similarly, proteinbased assays used to detect changes in expression of proteinscorresponding to the malignancy associated response signatures havevarying sensitivities. Thus, the degree to which increased expression isconsidered significant can vary depending on the nature of thetechniques used to measure expression, and one of ordinary skill in theart will understand this and choose a suitable platform and statisticalanalysis when detecting expression of the signature biomarkers, usingthe methods and assays described herein.

Similarly, the terms “decrease,” “reduced,” “reduction,” or “decrease”in connection with expression of the malignancy associated responsesignature markers are all used herein generally to refer to a decreaseby a statistically significant amount. However, for avoidance of doubt,“reduced”, “reduction” or “decrease” or “inhibit” means a decrease by atleast 10% as compared to a reference level, for example a decrease by atleast about 20%, or at least about 30%, or at least about 40%, or atleast about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90% or up to and including a 100%decrease (e.g. absent level or non-detectable level as compared to areference sample), or any decrease between 10-100% as compared to areference level.

The results described herein revealed that the 154 genes found in thesiRNA lethality screen to be selectively required for survival of cellswith malignant potential or cancer initiating cells, fall into aspecific groups of pathways, such as those shown in FIG. 3. Thus, insome aspects and embodiments, a malignancy associated response signaturecan comprise or consist essentially of specific subsets of the 154biomarkers all related to a specific cellular function or pathway.Examples of such pathways or functions include, but are not limited to,proteasome degradation, gene expression, DNA binding, DNA repair, RNAsplicing, apoptosis, cell adhesion, cell signaling, molecular transport,inflammation, mitosis, G1/S transition, metabolism, and the like. Bydetermining expression patterns of genes belonging to particularpathways, targeted therapies can be selected, as described elsewhereherein. For example, in some aspects and embodiments described herein,malignancy associated response signature biomarkers for expressionanalysis include subunits of the ubiquitin-proteasome complex listed inTable 3 including, but not limited to, PSMA1 (SEQ ID NO: 2), PSMA2 (SEQID NO: 33), PSMA3 (SEQ ID NO: 7), PSMB4 (SEQ ID NO: 1), PSMC1 (SEQ IDNO: 38), PSMC3 (SEQ ID NO: 4), PSMD2 (SEQ ID NO: 15), PSMD7 (SEQ ID NO:34), PSMD14 (SEQ ID NO: 16), UBL5 (SEQ ID NO: 29), NEDD1 (SEQ ID NO:149), NEDD8 (SEQ ID NO: 76), ANAPC2 (SEQ ID NO: 68), and ANAPC4 (SEQ IDNO: 101).

Accordingly, in some aspects, provided herein are malignancy associatedresponse signatures for a cancer-initiating cell susceptible toproteasomal gene inhibition comprising or consisting essentially of anexpression pattern of a set of at least 3 biomarkers set forth in SEQ IDNOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149, whereat least 3 of the 15 biomarkers have increased expression compared to areference value. The number of biomarkers selected for expressionanalysis can be at least 4, at least 5, at least 6, at least 7, at least8, least 9, at least 10, at least 11, at least 12, at least 13, least14, or include all 15.

In some aspects, provided herein are malignancy associated responsesignature for a cancer-initiating cell susceptible to mitosis geneinhibition comprising or consisting essentially of an expression patternof a set of at least 3 biomarkers set forth in SEQ ID NOs: 9, 17, 22,23, 31, 37, 35, 52, 68, 80, 101, 124, 130, and 137, where at least 3 ofthe 14 biomarkers have increased expression compared to a referencevalue. The number of biomarkers selected for expression analysis can beat least 4, at least 5, at least 6, at least 7, at least 8, least 9, atleast 10, at least 11, at least 12, at least 13, or include all 14.

In some aspects, provided herein are malignancy associated responsesignatures for a cancer-initiating cell susceptible to RNA splicing geneinhibition comprising or consisting essentially of an expression patternof a set of at least 3 biomarkers set forth in SEQ ID NOs: 5, 10, 11,18, 20, 26, 36, 41, 57, 61, 123, 127, and 151, where at least 3 of the13 biomarkers have increased expression compared to a reference value.The number of biomarkers selected for expression analysis can be atleast 4, at least 5, at least 6, at least 7, at least 8, least 9, atleast 10, at least 11, at least 12, or include all 13.

In some aspects, provided herein are malignancy associated responsesignatures for a cancer-initiating cell susceptible to moleculartransport gene inhibition comprising or consisting essentially of anexpression pattern of a set of at least 3 biomarkers set forth in SEQ IDNOs: 12, 13, 32, 48, 51, 56, 64, 91, 96, 98, 108, 121, and 147, where atleast 3 of the 14 biomarkers have increased expression compared to areference value. The number of biomarkers selected for expressionanalysis can be at least 4, at least 5, at least 6, at least 7, at least8, least 9, at least 10, at least 11, at least 12, at least 13, orinclude all 14.

In some aspects, provided herein are malignancy associated responsesignatures for a cancer-initiating cell susceptible to metabolic geneinhibition comprising or consisting essentially of an expression patternof a set of at least 3 biomarkers of SEQ ID NOs: 46, 49, 65, 69, 72, 78,104, 107, 112, 126, 138, 141, and 153, where at least 3 of the 13biomarkers have increased expression compared to a reference value. Thenumber of biomarkers selected for expression analysis can be at least 4,at least 5, at least 6, at least 7, at least 8, least 9, at least 10, atleast 11, at least 12, or include all 13.

Expression levels of a malignancy associated response signaturebiomarker can be determined by comparison to a suitable reference value,such that each of the expression levels of the at least two or more,e.g., 5 or more, 10 or more, malignancy associated response signaturebiomarker genes measured in a sample, such as a cellular sample, arecompared to a specific reference level that acts a standard ofcomparison. The reference level is obtained or measured in a referencebiological sample, such as a reference sample obtained from anage-matched normal control (e.g., an age-matched subject not having abreast cancer, or having a non-malignant or high prognosis breastcancer), or a reference sample from the same subject not from the tumoror cancer site, e.g., healthy breast tissue cells. A “reference value”is thus typically a predetermined reference level, such as an average ormedian of expression levels of each of the, for example, at least 2 ofthe 154 malignancy associated response signature associated biomarkers(SEQ ID NOs: 1-154) obtained from, for example, biological samples froma population of healthy subjects that are in the chronological age groupmatched with the chronological age of the tested subject. In someembodiments, the reference biological samples can also be gendermatched. For example, as explained herein, malignancy associatedresponse signature expression levels in a sample can be assessedrelative to normal breast tissue from the same subject or from a samplefrom another subject or from a repository of normal subject samples. Ifthe expression level of a malignancy associated response signaturebiomarker is greater or less than that of the reference or the averageexpression level of, for example, non-basal-like breast tumors of aparticular type, the malignancy associated response signature biomarkerexpression is said to be “increased” or “decreased,” respectively, asthose terms are defined herein. Exemplary analytical methods forclassifying expression of a malignancy associated response signaturebiomarker, determining a malignancy associated response signaturestatus, and scoring of a sample for expression of a malignancyassociated response signature biomarker are explained in detail herein.

Accordingly, in some embodiments of the malignancy associated responsesignatures described herein, an increased expression over a referencevalue is at least about a 1.5-fold increase, at least about a 1.6-foldincrease, at least about a 1.7-fold increase, at least about a 1.8-foldincrease, at least about a 1.9-fold increase, at least about a 2-foldincrease, at least about a 3-fold increase, at least about a 4-foldincrease, at least about a 5-fold increase, at least about a 10-foldincrease, any increase between 1.5-fold and 10-fold, at least about a25-fold increase, or greater as compared to a reference level. In someembodiments, an increase is at least about 1.8-fold increase over areference value.

The malignancy associated response signatures and assays and methodsthereof described herein are useful for identifying cancer-initiatingcells and classifying cancers as having poor prognosis, high malignantpotential, or comprising cancer-initiating cells, for example.“Cancer-initiating cells,” which also include cancer stem cells, referto cells that are neoplastic and can undergo self-renewal as well asabnormal proliferation and differentiation. Functional features ofcancer-initiating cells are that they are tumorigenic, i.e., causecancers or tumors. Such cancer-initiating cells can give rise toadditional such neoplastic cells by self-renewal, and they can give riseto non-tumorigenic neoplastic cells. Without being bound to anyparticular theory, cancer initiating cells contribute to the developmentof metastatic cancer and relapses. The unique malignancy associatedresponse signature biomarkers, described herein, permit specifictherapies targeted at cancer-initiating cells.

Accordingly, in some embodiments of the malignancy associated responsesignatures, the cancer-initiating cell is a breast cancer-initiatingcell. In some embodiments of the malignancy associated responsesignatures, the cancer-initiating cell is a triple-negative breastcancer-initiating cell. In some embodiments of the malignancy associatedresponse signatures, the cancer-initiating cell is a Luminal B breastcancer-initiating cell. In some embodiments of the malignancy associatedresponse signatures, the cancer-initiating cell is an epithelial breastcancer-initiating cell.

A “cancer” or “tumor” as used herein refers to an uncontrolled growth ofcells which interferes with the normal functioning of the bodily organsand systems. A subject that has a cancer or a tumor is a subject havingobjectively measurable cancer cells present in the subject's body.Included in this definition are benign and malignant cancers, as well asdormant tumors or micrometastatses. Cancers which migrate from theiroriginal location and seed vital organs can eventually lead to the deathof the subject through the functional deterioration of the affectedorgans. Hemopoietic cancers, such as leukemia, are able to out-competethe normal hemopoietic compartments in a subject, thereby leading tohemopoietic failure (in the form of anemia, thrombocytopenia andneutropenia) ultimately causing death.

By “metastasis” is meant the spread of cancer from its primary site toother places in the body. Cancer cells can break away from a primarytumor, penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. Metastasis can be local or distant. Metastasis isa sequential process, contingent on tumor cells breaking off from theprimary tumor, traveling through the bloodstream, and stopping at adistant site. At the new site, the cells establish a blood supply andcan grow to form a life-threatening mass. Both stimulatory andinhibitory molecular pathways within the tumor cell regulate thisbehavior, and interactions between the tumor cell and host cells in thedistant site are also significant.

Metastases are most often detected through the sole or combined use ofmagnetic resonance imaging (MRI) scans, computed tomography (CT) scans,blood and platelet counts, liver function studies, chest X-rays and bonescans, in addition to the monitoring of specific symptoms.

Examples of cancer for use in the methods of diagnosis, prognosis, andtreatment described herein, include but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia. More particular examples ofsuch cancers include, but are not limited to, breast cancer (including,e.g., triple negative breast cancers, basal-like breast cancers, LuminalB breast cancers); basal cell carcinoma, biliary tract cancer; bladdercancer; bone cancer; brain and CNS cancer; cancer of the peritoneum;cervical cancer; choriocarcinoma; colon and rectum cancer; connectivetissue cancer; cancer of the digestive system; endometrial cancer;esophageal cancer; eye cancer; cancer of the head and neck; gastriccancer (including gastrointestinal cancer); glioblastoma; hepaticcarcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer;larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung); lymphoma including Hodgkin's andnon-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavitycancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer;pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma;rectal cancer; cancer of the respiratory system; salivary glandcarcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer;testicular cancer; thyroid cancer; uterine or endometrial cancer; cancerof the urinary system; vulval cancer; as well as other carcinomas andsarcomas; as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome.

Also provided herein are assays and methods for classifying cancers in asubject based on expression analyses of various combinations of thebiomarkers of the malignancy associated response signatures describedherein. By classifying a cancer as having poor prognosis or malignantpotential, and identifying pathways that are associated with poorprognosis or malignant potential, specific genes and biomarkers, such asthose described herein, can be targeted, and specific therapies can beadministered to a subject. A cancer or tumor, such as a breast cancer,can be classified as highly malignant or having poor prognosis byassessing and determining increased expression levels of at least two ormore, preferably at least 10, of the 154 malignancy associated responsesignature genes or biomarkers listed in Table 3 (SEQ ID NOs: 1-154) in abiological sample in any combination, or in combination with any otherprognostic biomarker known to one of skill in the art. Preferably,expression levels of at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 23, at least25, at least 30, at least 35, at least 40, at least 45, at least 50, atleast 100, at least 125, or more of the 154 malignancy associatedresponse signatures genes described herein are assessed. It is wellwithin the ability of one skilled in the art, using the teachingsprovided herein, to identify additional genes having prognostic orpredictive value for use with the assays and methods described herein.

Accordingly, in some aspects, provided herein are methods of classifyinga cancer in a subject in need thereof, such methods comprising:

-   -   a. assaying expression of ten or more of the 154 malignancy        associated response signature biomarkers of SEQ ID NOs: 1-154 in        a biological sample obtained from the subject having a cancer,        and    -   b. comparing the expression of the ten or more of the 154        malignancy associated response signature biomarkers of SEQ ID        NOs: 1-154 in the biological sample obtained from the subject        having a cancer with a reference value, such that increased        expression of 1.8-fold or greater of at least ten of the        biomarkers in the biological sample obtained from the subject        relative to the reference value indicates that the cancer is        classified as having a poor prognosis or being a malignant        cancer, and absence of increased expression of 1.8-fold or        greater of at least ten of the biomarkers relative to the        reference value indicates that the cancer does not have poor        prognosis or is not a malignant cancer.

In some aspects, provided herein are methods of classifying a cancer ina subject in need thereof, such methods comprising:

-   -   a. assaying expression of at least five biomarkers set forth in        SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72,        77, 78, 84, 91, 93, and 151 in a biological sample obtained from        the subject having a cancer, and    -   b. comparing the expression of the five or more of the 23        malignancy associated response signature biomarkers in the        biological sample obtained from the subject having a cancer with        a reference value, wherein increased expression of 1.8-fold or        greater of at least ten of the biomarkers in the biological        sample obtained from the subject relative to the reference value        indicates that the cancer is classified as having a poor        prognosis or being a malignant cancer, and absence of increased        expression of 1.8-fold or greater of at least ten of the        biomarkers relative to the reference value indicates that the        cancer does not have poor prognosis or is not a malignant        cancer.

In some aspects, provided herein are methods of classifying a cancer ina subject in need thereof, the method comprising:

-   -   a. assaying expression of at least three malignancy associated        response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4,        7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149 in a        biological sample obtained from the subject having a cancer, and    -   b. comparing the expression of the at least three malignancy        associated response signature biomarkers in the biological        sample obtained from the subject having a cancer with a        reference value, wherein increased expression of 1.8-fold or        greater of at least three of the biomarkers in the biological        sample obtained from the subject relative to the reference value        indicates that the cancer is classified as having a poor        prognosis or being a malignant cancer, and absence of increased        expression of 1.8-fold or greater of at least three of the        biomarkers relative to the reference value indicates that the        cancer does not have poor prognosis or is not a malignant        cancer.

Also provided herein, in some aspects, are assays for determining cancerprognosis in a subject. Such assays comprise the steps of:

-   -   a. measuring expression of ten or more of the 154 malignancy        associated response signature biomarkers set forth in SEQ ID        NOs: 1-154 in a biological sample obtained from a subject having        cancer, and    -   b. comparing the expression of the ten or more of the 154        malignancy associated response signature biomarkers set forth in        SEQ ID NOs: 1-154 in the biological sample obtained from the        subject having a cancer with a reference value, wherein        increased expression of at least ten of the measured biomarkers        in the biological sample obtained from the subject relative to        the reference value diagnoses the patient as having a poor        prognosis or malignant cancer, and absence of increased        expression of at least ten of the measured biomarkers relative        to the reference value diagnoses the patient as not having a        poor prognosis or malignant cancer.

In some embodiments of these methods and assays, the cancer is a breastcancer. In some embodiments of these methods and assays, the cancer is atriple-negative breast cancer. In some embodiments of these methods andassays, the cancer is a Luminal B breast cancer. In some embodiments ofthese methods and assays, the cancer is an epithelial breast cancer.

In some embodiments of these assays and methods, increased expression ofthe two or more of the 154 malignancy associated response signaturebiomarkers, or a subset thereof, in the biological sample classifies thecancer or determines a prognosis for the subject having the cancer. Insome such embodiments, the prognosis comprises relative survival rate,relative risk of metastasis, treatment option, or any combinationthereof.

As demonstrated herein, the described malignancy associated responsesignature biomarker genes have prognostic value. The term “prognosis,”as used herein, refers to prediction of a cancer attributableprogression. Prognosis includes, but is not limited to the survival rateand risk of recurrence, metastatic spread, treatment resistance, andlocal-regional failure. In the examples described herein, increasedexpression and enrichment of malignancy associated response signaturebiomarkers in luminal B subtype breast tumors was found to correlatewith decreased survival. In breast cancer patients, enriched expressionof malignancy associated response signature genes was found to correlatewith increased risk and onset of metastasis, and decreasedmetastasis-free survival. Accordingly, expression of malignancyassociated response signature biomarkers in a tumor sample can be usedclinically as both a predictive and prognostic marker to provideinformation concerning appropriate treatment modalities and likelytreatment outcome. The expression of malignancy associated responsesignature biomarkers can be used in conjunction with other prognosticfactors, in some embodiments, to allow a clinician to predict theoutcome of a cancer treatment. The stratification of patients based onenriched expression of malignancy associated response signaturebiomarkers can be used to determine a treatment protocol for thepatients. For example, patients can be divided into those who have a lowrisk for metastasis and can be spared specific therapies, those withhigh risk for metastasis who are likely to benefit from standardtreatments, and those with a high risk for metastasis and who areresistant to standard treatments. Such stratification permits tailoringof patient treatment protocols to the individual patient.

The terms “subject,” “patient,” and “individual” are usedinterchangeably herein, and refer to an animal, for example a human. Fortreatment of those disease states which are specific for a specificanimal such as a human subject, the term “subject” refers to thatspecific animal. The terms ‘non-human animals’ and ‘non-human mammals’are used interchangeably herein, and include mammals such as rats, mice,rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term“subject” also encompasses any vertebrate including but not limited tomammals, reptiles, amphibians and fish. In some embodiments of theaspects described herein, a subject refers to a human subject having abreast cancer or at increased risk for a breast cancer. A subject thathas a breast cancer or a tumor is a subject having objectivelymeasurable breast cancer cells present in the subject's body.

As used herein, the term “biological sample” or “subject sample” or“sample” refers to a cell or population of cells or a quantity of tissueor fluid obtained from a subject. Most often, the sample has beenremoved from a subject, but the term “biological sample” can also referto cells or tissue analyzed in vivo, i.e. without removal from thesubject. A biological sample or tissue sample includes, but is notlimited to, blood, plasma, serum, lymph fluid, bone marrow, tumorbiopsy, urine, stool, sputum, cerebrospinal fluid, pleural fluid, nippleaspirates, lymph fluid, the external sections of the skin, lung tissue,adipose tissue, connective tissue, sub-epithelial tissue, epithelialtissue, liver tissue, kidney tissue, uterine tissue, respiratorytissues, breast tissue, gastrointestinal tissue, and genitourinary tracttissue, tears, saliva, milk, cells (including, but not limited to, bloodcells), biopsies, scrapes (e.g., buccal scrapes), tumors, organs, andalso samples of an in vitro cell culture constituent. Often, a“biological sample” will contain cells from the subject, but the termcan also refer to non-cellular biological material, such as non-cellularfractions of blood, saliva, or urine. In some embodiments, the sample isfrom a resection, or core needle biopsy of a primary or metastatictumor, such as a breast tumor, or a cell block from pleural fluid. Insome embodiments, fine needle aspirate samples are used. Samples can beparaffin-embedded or frozen tissue.

In another aspect, provided herein are methods of determining the typeor prognosis of a tumor from a patient, the methods comprising detectingexpression of ten or more of the 154 triple negative breast cancer genesignature genes of Table 3 (SEQ ID NOs: 1-154) in a biological samplefrom a subject affected with or having a tumor, wherein increasedexpression of the genes relative to a reference sample indicates thatthe tumor is a poor prognosis tumor, such as a triple-negative tumor orbasal-like breast tumor, and absence of increased expression of thegenes relative to the reference indicates that the tumor is not a poorprognosis tumor.

In other aspects, provided herein are methods of determining the type ofa breast tumor from a patient comprising detecting expression of ten ormore of the 154 triple negative breast cancer gene signature genes ofTable 3 (SEQ ID NOs: 1-154) in a biological sample from a subjectaffected with or having a triple-negative breast cancer, whereinincreased expression of the genes relative to a reference sampleindicates that the triple-negative breast cancer is a basal-like breasttumor or poor prognosis luminal B breast tumor, and absence of increasedexpression of the genes relative to the reference indicates that thetumor is not a basal-like breast tumor or a poor prognosis luminal Bbreast tumor.

In some embodiments of these aspects and all such aspects describedherein, the methods and assays further comprise the step ofadministering at least one proteasome inhibitor to the subject havingbeen determined to carry a poor prognosis tumor, a triple-negativetumor, a basal-like breast tumor, or a poor prognosis luminal B breasttumor. In some embodiments, the proteasome inhibitor is bortezomib. Insome embodiments, the proteasome inhibitor is an siRNA or antisense RNAagent.

In some embodiments of these aspects and all such aspects describedherein, the method further comprises the step of administering at leastone histone deactylase inhibitor to the subject having been determinedto carry a poor prognosis tumor, a triple-negative breast cancer, abasal-like breast tumor, or a poor prognosis luminal B breast tumor. Insome such embodiments, the histone deactylase inhibitor is TrichostatinA. In some such embodiments, the histone deactylase inhibitor isVorinostat. In some embodiments, the histone deactylase inhibitor is ansiRNA or antisense RNA agent.

In some embodiments of these aspects and all such aspects describedherein, the method further comprises the step of administering at leastone glycolysis inhibitor to the subject having been determined to carrya poor prognosis tumor, a triple-negative breast cancer, a basal-likebreast tumor, or a poor prognosis luminal B breast tumor. In some suchembodiments, the glycolysis inhibitor is 3-bromo-pyruvic acid (BRPA). Insome embodiments, the glycolysis inhibitor is an siRNA or antisense RNAagent.

In various embodiments of the aspects described herein, expressionlevels of the TGS biomarkers in a biological sample can be evaluated byany suitable means known to one of skill in the art. For example, insome embodiments, expression can be evaluated using DNA microarrays.Alternatively, in other embodiments, RNA transcripts can be measuredusing real time PCR, or protein products can be detected using suitableantibodies. Methods of determining expression levels of genes by theseand other methods are known in the art.

In some embodiments of these aspects and all such aspects describedherein, the expression levels of the at least two or more, e.g., 10 ormore, malignancy associated response signature biomarker genes measuredin a biological sample are compared to a reference level, or a referencebiological sample, such as biological sample obtained from anage-matched normal control (e.g., an age-matched subject not having abreast cancer, or having a non-malignant or high prognosis breastcancer), or a biological sample from the same subject not from the tumoror cancer site, e.g., healthy breast tissue cells. For diagnosing ordetermining a prognosis of a subject having a breast cancer in a subjectusing the methods and systems as disclosed herein, a “reference value”is typically a predetermined reference level, such as an average ormedian of expression levels of the at least 2 of the 154 malignancyassociated response signature biomarkers (SEQ ID NOs: 1-154) obtainedfrom biological samples from a population of healthy subjects that arein the chronological age group matched with the chronological age of thetested subject. As indicated earlier, in some situations, the referencebiological samples can also be gender matched.

Also provided herein are assays for determining or identifying whether asample from a subject, such as a biopsy sample, comprises cancerinitiating cells. Such assays are based, in part, on the high-throughputsiRNA lethality screen described herein, where different cancer typeswere assessed for differential susceptibility to gene inhibition using alibrary of different siRNA sequences targeted to different genes.

Such assays comprise the steps of: (a) dividing a cell culture grownfrom a biopsy obtained from a subject having cancer into at least 5separate cultures; (b) exposing each of the at least 5 separate culturesto a different inhibitory agent, such that each of the differentinhibitory agent specifically inhibits a different malignancy associatedresponse signature biomarker set forth in SEQ ID NOs: 1-154; (c) growingeach of the at least 5 separate cell cultures of step (b) for at least12 hours; and (d) measuring viability of the cells from each of thecultures of step (c), such that if the total viability of the cells inat least 60% of the cultures is decreased by at least 25%, then thebiopsy obtained from the subject comprises cancer-initiating cells.

Viability, as used herein refers, to the number of living cells presentin a culture at a given timepoint. Viability can be measured using anyof the assays set forth herein, such as those described in the Examplessection. Exemplary viability assays include Trypan Blue staining, flowcytometric analyses of annexin V, propidium iodide staining and thelike.

Cells can be cultured according to standard methods known to one ofskill in the art, and the duration of culture can vary. Preferably aculture for identifying whether a sample comprises cancer-initiatingcells is grown for at least 6 hours, at least 12 hours, at least 18hours, at least 24 hours, at least 30 hours, at least 36 hours, at least42 hours, at least 48 hours, at least 54 hours, at least 60 hours, atleast 66 hours, at least 72 hours, at least 84 hours, at least 96 hours,at least 108 hours, at least 120 hours, or more. As understood by one ofordinary skill in the art, culture itself can cause loss in viability ofcells, due, for example, to overcrowding in a dish or well, and thus thecultures should be grown for long enough to identify and measure anyeffect(s) on the cells from the inhibitory agent to which the cells havebeen exposed, but not so long as to mask any such effects due, forexample, to overcrowding.

In some embodiments of these assays and all such assays describedherein, the inhibitory agents are selected from siRNA agents, antisenseRNA agents, or small molecules, that specifically inhibit any of thecancer gene signature biomarkers set forth in SEQ ID NOs: 1-154.

In some embodiments of these assays and all such assays describedherein, the inhibitory agents specifically inhibit any of the malignancyassociated response signature biomarkers set forth in SEQ ID NOs: 1-4,6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and151.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of themalignancy associated response signature biomarkers set forth in SEQ IDNOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91,93, and 151.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of theproteasomal malignancy associated response signature biomarkers setforth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76,and 149.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of themitosis cancer gene signature biomarkers set forth in SEQ ID NOs: 9, 17,22, 23, 31, 37, 35, 52, 68, 80, 101, 124, 130, and 137.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of theRNA splicing malignancy associated response signature biomarkers setforth in SEQ ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57, 61, 123, 127,and 151.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of themolecular transport malignancy associated response signature biomarkersset forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56, 64, 91, 96, 98, 108,121, and 147.

In some embodiments of these assays and all such assays describedherein, at least one inhibitory agent specifically inhibits any of themetabolic malignancy associated response signature biomarkers set forthin SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and153.

In some embodiments of the aspects described herein, the assays andmethods for classifying a cancer, determining whether a sample comprisescancer-initiating cells, determining or diagnosing the type of a canceror tumor from a patient, providing a prognosis to a cancer patient, ortreating a cancer patient, further comprise the step of selecting oridentifying the subject having the cancer. In such embodiments, asubject is identified as having cancer by objective determination of thepresence of cancer cells or a tumor in the subject's body by one ofskill in the art. Such objective determinations can be performed throughthe sole or combined use of manual examination, tissue biopsies, bloodand platelet cell counts, mammograms, ultrasound, urine analyses,magnetic resonance imaging (MRI) scans, computed tomography (CT) scans,liver function studies, chest X-rays and bone scans in addition to themonitoring of specific symptoms associated with the cancer.

Expression levels of a malignancy associated response signaturebiomarker can be determined by comparison to a suitable reference. Forexample, as explained herein, malignancy associated response signaturebiomarker expression levels in a sample can be assessed relative tonormal breast tissue from the same subject or from a sample from anothersubject or from a repository of normal subject samples. Alternatively,the relative expression level of a malignancy associated responsesignature gene in a particular tumor can be determined with reference tothe expression levels of the same gene in for example, non-basal-likebreast tumors, e.g., HER2, luminal, or claudin-low breast cancers. Ifthe expression level of a malignancy associated response signature geneis greater or less than that of the reference or the average expressionlevel of non-basal-like breast tumors of a particular type, themalignancy associated response signature gene expression is said to be“increased” or “decreased,” respectively, as those terms are definedherein.

In other aspects, provided herein are methods for diagnostic orprognostic analysis of a cancer, such as a breast cancer, in a subjectcomprising the step of measuring expression of at least 10 of the 154genes of a malignancy associated response signature (SEQ ID NOs: 1-154)for a cancer-initiating cell in a biological sample, wherein said triplenegative breast cancer gene signature comprises an expression pattern ofa set of 10-154 biomarker genes set forth in Table 3 (SEQ ID NOs:1-154), and wherein an increase of expression of at least 10 of thebiomarker genes is indicative of poor prognosis of the cancer in thesubject.

In some embodiments of this aspect, the expression of the at least 10 ofthe 154 biomarkers of the malignancy associated response signature inthe biological sample determines a prognosis for the subject having thecancer. In some such embodiments, the prognosis comprises relativesurvival rate, relative risk of metastasis, treatment option, or anycombination thereof.

In further embodiments of the prognostic methods described herein, therelative survival rate of a patient can be predicted by determining theexpression of at least one, but preferably at least 10, malignancyassociated response signature biomarkers in a tumor sample derived froma patient. Survival in cancer patients is often quantified as a“relative cancer survival rate.” The relative cancer survival rate isthe percentage of patients who survive a certain type of cancer for aspecified amount of time. Cancer survival rates are often given in 5, 10or 20 year survival rates. As seen in the examples described herein,enriched expression of malignancy associated response signaturebiomarkers can predict differential survival rates of patients withsubgroups of Luminal B cancer, and metastasis-free survival rates inprimary breast cancer samples.

Pathway Inhibitors & Therapeutic Methods

The various combinations of malignancy associated response signaturebiomarkers identified in the screens described herein provide noveltherapeutic targets and pathways, such as the pathways highlighted inFIG. 3, e.g., ubiquitin-proteosome pathway, histone deactylase pathway,metabolic pathway, for treatment of subjects having cancers classifiedas malignant or poor prognosis, or having cancer-initiating cells, suchas, for example, triple negative breast cancers.

Accordingly, in some embodiments, if a cancer is classified as having apoor prognosis or being a malignant cancer, or comprisingcancer-initiating cells using, for example, the assays and methods forclassifying cancers provided herein, at least one proteasome inhibitoris administered to the subject.

For example, in some embodiments, if the cancer is classified as havinga poor prognosis or being a malignant cancer, or comprisingcancer-initiating cells using, for example, the assays and methods forclassifying cancers provided herein, such that at least 10 of the 154genes of a malignancy associated response signature (SEQ ID NOs: 1-154)for a cancer-initiating cell in a biological sample have increasedexpression over a reference value, then at least one proteasomeinhibitor is administered to the subject. In some such embodiments, if,in addition to the requirement that at least 10 of the 154 genes of amalignancy associated response signature (SEQ ID NOs: 1-154) areincreased relative to reference value, at least two of the ten or moregenes having increased expression are required to be proteasomalmalignancy associated response signature biomarkers of SEQ ID NOs: 1, 2,4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149, then at least oneproteasome inhibitor is administered to the subject.

Similarly, in some embodiments, if the cancer is classified as having apoor prognosis or being a malignant cancer, or comprisingcancer-initiating cells using, for example, the assays and methods forclassifying cancers provided herein, such that at least five of thebiomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35,37, 38, 66, 72, 77, 78, 84, 91, 93, and 151 have increased expressionrelative to a reference value, then at least one proteasome inhibitor isadministered to the subject. In some such embodiments, if, in additionto the requirement that at least five of the 23 genes of the malignancyassociated response signature are increased relative to reference value,at least one of the five or more genes having increased expression arerequired to be proteasomal malignancy associated response signaturebiomarkers of SEQ ID NOs: 1, 2, 4, 7, 29, 33, 34, and 38, then at leastone proteasome inhibitor is administered to the subject.

The term “proteosome inhibitor,” as used herein, relates to a compoundor an agent which targets, decreases or inhibits one or more componentsof the proteosome or proteosomal pathway. The proteasome is anintracellular structure which is a multicatalytic proteinase which is ahighly conserved. Proteasomes are responsible for the ATP-dependentproteolysis of many proteins involved in important regulatory cellularprocesses. Thus, the proteosome is a regulatory element in cell growthand differentiation. Several steps are involved in protein degradationvia the proteasome or “ubiquitin-proteasome” pathway. Initially, aprotein is marked for destruction with a chain of small polypeptidesknown as ubiquitin. Ubiquitinylation guides the protein into theproteosome's enclosed proteolytic chamber. Three enzymatic activities,E1, E2, and E3, are required for ubiquitinylation. The ATP-dependent E1enzyme activates ubiquitin and links it to the ubiquitin-conjugatingenzyme, E2. The E3 enzyme, an ubiquitin ligase, then links the ubiquitinmolecule to the protein. This process is repeated until the designatedpolypeptide trails a long chain of ubiquitin moieties and the proteasomefinally degrades the protein into small fragments. Theubiquitin-proteasome pathway is responsible for the degradation of 90%of all abnormal, misfolded proteins and all of the short-lived,regulatory proteins in the cell.

Examples of targets of a proteosome inhibitor include, but are notlimited to, O(2)(−)-generating NADPH oxidase, NF-κB farnesyltransferase,and geranylgeranyltransferase I.

A variety of inhibitors of the proteasome complex have been reported,e.g., Dick, et al., Biochem. 30: 2725 (1991); Goldberg, et al., Nature357: 375 (1992); Goldberg, Eur. J. Biochem. 203: 9 (1992); Orlowski,Biochem. 29: 10289 (1989); Rivett, et al., Archs. Biochem. Biophys. 218:1 (1989); Rivett, et al., J. Biol. Chem. 264: 12, 215 (1989); Tanaka, etal., New Biol. 4: 1 (1992). Proteasome inhibitors are also discussed inU.S. Pat. No. 5,693,617, the disclosure of which is incorporated hereinby reference. In addition to antibiotic inhibitors originally isolatedfrom actinomycetes, a variety of peptide aldehydes have beensynthesized, such as the inhibitors of chymotrypsin-like proteasesdescribed by Siman et al. (WO91/13904).

Specific proteasome inhibitors fall into five classes distinguished bythe pharmacophore that interacts with the active site threonine in theproteasome: peptide aldehydes such as CEP1612 and MG132, peptideboronates such as bortezomib, peptide vinyl sulfones, peptideepoxyketones and β-lactone inhibitors such as lactocystin. In someembodiments of the aspects and embodiments provided herein, a proteasomeinhibitor is bortezomib.

Examples of proteosome inhibitors or analogues thereof contemplated foruse in the methods described herein include, but are not limited to,aclacinomycin A; MG-132; gliotoxin; PS-341; MLN 341; peptide aldehydes,e.g., N-acetyl-leucinyl-leucynil-norleucynal, N-acetyl-leucinylleucynil-methional, carbobenzoxyl-leucinyl-leucynil-norvalinal,carbobenzoxyl-leucinyl-leucynil-leucynal, lactacystine, b-lactone;boronic acid peptides; ubiquitin ligase inhibitors; PS-519(1R-[1S,4R,5S]]-1-(1-hydroxy-2-methylpropyl)-4-propyl-6-oxa-2-azabicyclo[-3.2.1.]heptane-3,7-dione);clasto-lactacystin beta-lactone; lactacystin; epoxomicin; CVT634(-5-methoxy-1-indanone-3-acetyl-leucyl-D-leucyl-1-indanylamide); TMC96((3-methylbutanoyl-L-threonineN-(1-(2-(hydroxymethyl)-oxiran-2-ylcarbonyl)-3-methylbut-3enyl)amide);MG-115; CEP161; cyclosporin A; deoxyspergualin; bortezomib and/orcompounds having structure similar to that of bortezomib; and Velcade.Proteasome inhibitors having structure similar to bortezomib includethose compounds disclosed in U.S. Pat. Nos. 7,119,080; 6,747,150;6,617,317; 6,548,668; 6,465,433; 6,297,217; 6,083,903; 5,780,454;7,422,830; 7,109,323; 6,958,319; 6,713,446; and 6,699,835, the contentsof each of which are herein incorporated by reference in theirentireties.

In addition to the proteasome protease inhibitors, theubiquitin-proteasome pathway can be blocked by inhibitors of thefacilitating enzymes Ubiquitin-activating enzyme (E1),ubiquitin-conjugating enzyme (E2), and ubiquitin-ligases (E3 enzymes).E1 inhibitors have been identified such as himeic acid A (Tsukamoto, etal. 2005, Bioorgan Med Chem Lett 15(1):191-194. Other methods known inthe art, such as RNA mediated silencing or inhibition, can also be usedto reduce or eliminate the activities of specificubiquitinylation-related enzymes.

MCL1 (SEQ ID NO: 134) is a myeloid cell leukemia protein of the Bcl-2family of proteins. Two alternatively spliced transcripts encodingdistinct isoforms have been identified. The longer gene product(isoform 1) enhances cell survival by inhibiting apoptosis while thealternatively spliced shorter gene product (isoform 2) promotesapoptosis and is death-inducing. The results described herein identifyMCL-1 as a malignancy associated response signature biomarker, anddemonstrate that inhibition of MCL-1 has the same lethal effects asproteasome inhibition in poor prognosis cancer cells, such astriple-negative breast cancer cells. Accordingly, in some embodiments, aproteasome inhibitor is chosen that specifically inhibits MCL-1 of SEQID NO: 134 for use with the assays and methods described herein.

In some embodiments, the at least one proteasome inhibitor specificallyinhibits any of the proteasomal malignancy associated response signaturebiomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34,38, 60, 74, 76, and 149. In some embodiments, the at least oneproteasome inhibitor specifically inhibits the malignancy associatedresponse signature biomarker set forth in SEQ ID NO: 134 (MCL-1).

In some embodiments, the at least one proteasome inhibitor is an siRNAor antisense RNA agent.

In some embodiments, the at least one proteasome inhibitor isbortezomib.

In some embodiments of the methods and assays described herein, if acancer is classified as having a poor prognosis or being a malignantcancer, at least one histone deacetylase inhibitor is administered tothe subject having cancer. In some such embodiments, the at least onehistone deacetylase inhibitor is an siRNA or antisense RNA agent. Insome such embodiments, the at least one histone deacetylase inhibitor istrichostatin A (TSA) or Vorinostat.

The terms “histone deacetylase inhibitor” or “inhibitor of histonedeacetylase,” as used herein, relate to a compound or an agent whichtargets, decreases, or inhibits the activity or expression of one ormore histone deacetylases or components of the histone deacetylasepathway. Inhibiting histone deacetylase enzymatic activity meansreducing the ability of a histone deacetylase to remove an acetyl groupfrom a histone or another protein substrate. Preferably, such inhibitionis specific, i.e., the histone deacetylase inhibitor reduces the abilityof a histone deacetylase to remove an acetyl group from a histone oranother protein substrate at a concentration that is lower than theconcentration of the inhibitor that is required to produce some other,unrelated biological effect. The terms “histone deacetylase” and “HDAC”are intended to refer to any one of a family of enzymes that removeacetyl groups from the E-amino groups of lysine residues at theN-terminus of a histone. Unless otherwise indicated by context, the term“histone” is meant to refer to any histone protein, including H1, H2A,H2B, H3, H4, and H5, from any species. Human HDAC proteins or geneproducts, include, but are not limited to, HDAC-1, HDAC-2, HDAC-3,HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10 and HDAC-11. Thehistone deacetylase can also be derived from a protozoal or fungalsource.

Any histone deacetylase inhibitor can be used in the context of thepresent invention. Histone deacetylase inhibitors from various chemicalclasses have been described, with four most important classes, namely(i) hydroxamic acid analogs, (ii) benzamide analogs, (iii) cyclicpeptides (generally tetrapeptides)/peptolides and (iv) fatty acidanalogs. Histone deacetylase inhibitors differ in their specificitiestowards the various histone deacetylases. These enzymes are divided intofour main classes according to their sequence homology and expressionpatterns. Histone deacetylase inhibitors differ in their specificitiestowards the four main classes of histone deacetylases. These enzymes aredivided into four classes according to their sequence homology andexpression patterns. Generally hydroxamic acid analogs are effective onclasses I, II, IV enzymes, benzamide analogs on class I and some also onclasses II, III and/or IV, cyclic peptides/peptolides on class I andfatty acid analogs on classes I and IL Brief overviews on histonedeacetylase inhibitors have recently been given by Smith and Workman(International Journal of Biochemistry & Cell Biology (2008)doi:10.1016/j.biocel.2008.09.008) and, in a broader context, by Szyf(Annu. Rev. Pharmacol. Toxicol. (2009) 49, 243-263).

Suitable examples of a histone deacetylase inhibitor for use in themethods described herein include, but are not limited to, trichostatin A(TSA), N′-hydroxy-N-phenyl-octanediamide (suberoylanilide hydroxamicacid, SAHA), pyroxamide, M-carboxycinnamic acid bishydroxamide (CBHA),trichostatin C, salicylihydroxamic acid (SBHA), azelaic bishydroxamicacid (ABHA), azelaic-1-hydroxamate-9-anilide (AAHA),6-(3-chlorophenylureido) carpoic hydroxamic acid (3C1-UCHA), oxamflatin,A-161906, scriptaid, PXD-101, LAQ-824, cyclic hydroxamic acid-containingpeptide (CHAP), ITF-2357, MW2796, MW2996, trapoxin A, FR901228 (FK 228or Depsipeptide), FR225497, apicidin, CHAP, HC-toxin, WF27082,chlamydocin, sodium butyrate, isovalerate, valerate, 4-phenylbutyrate(4-PBA), 4-phenylbutyrate sodium (PBS), arginine butyrate, propionate,butyramide, isobutyramide, phenylacetate, 3-bromopropionate, tributyrin,valproic acid, valproate, CI-994, MS-27-275 (MS-275 or SNDX-275),3′-amino derivative of MS-27-275, MGCD0103 or Depudecin, and SNDX-275. Anumber of histone deacetylase inhibitors are currently being clinicallytested and SAHA (VORINOSTAT™) has recently been approved by the FDA fortreatment of cutaneous T cell lymphoma.

In some embodiments of the methods and assays described herein, if acancer is classified as having a poor prognosis or being a malignantcancer, at least one glygolytic or metabolic inhibitor is administeredto the subject. In some such embodiments, the at least one glycolysisinhibitor is an siRNA or antisense RNA agent.

The terms “metabolic inhibitor,” “glycolysis inhibitor” or “glycolyticinhibitor,” as used herein, relates to a compound or an agent whichtargets, decreases, stops, or inhibits one or more components of theglycolytic or glycolysis pathway. Glycolysis refers to the metabolicpathway that converts glucose C6H12O6, into pyruvate, CH3COCOO—+H+. Thefree energy released in this process is used to form the high-energycompounds ATP (adenosine triphosphate) and NADH (reduced nicotinamideadenine dinucleotide). Glycolysis involves a sequence of ten reactionsinvolving ten intermediate compounds (one of the steps involves twointermediates). The intermediates provide entry points to glycolysis.For example, most monosaccharides, such as fructose, glucose, andgalactose, can be converted to one of these intermediates. Theintermediates can also be directly useful. For example, the intermediatedihydroxyacetone phosphate (DHAP) is a source of the glycerol thatcombines with fatty acids to form fat.

The most common type of glycolysis is the Embden-Meyerhof-Parnas pathway(EMP pathway). Glycolysis also refers to other pathways, such as theEntner-Doudoroff pathway and various heterofermentative andhomofermentative pathways. Suitable examples of a glycolysis inhibitorfor use in the methods described herein include, but are not limited to,3-bromo-pyruvic acid (bromoethylpyrruvate), Hypoglycin A,2-deoxy-D-glucose, dichloro-acetate, oxamate and other pyruvate analogs,staurosporine, oligomycin, 6-dichloro-1,6-dideoxy-2-deoxyglucose,2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG),2-fluor-2-deoxy-D-glucose (2FG), 2-deoxy-D-galactose, 3H-2-deoxyglucoseor analogs thereof. In addition, examples of 2-deoxyglucose compoundsuseful in the methods described herein include, but are not limited to,2-deoxy-D-glucose, 2-deoxy-L-glucose; 2-bromo-D-glucose,2-fluoro-D-glucose, 2-iodo-D-glucose, 6-fluoro-D-glucose,6-thio-D-glucose, 7-glucosyl fluoride, 3-fluoro-D-glucose,4-fluoro-D-glucose, 1-O-propyl ester of 2-deoxy-D-glucose, 1-O-tridecylester of 2-deoxy-D-glucose, 1-O-pentadecyl ester of 2-deoxy-D-glucose,3-O-propyl ester of 2-deoxy-D-glucose, 3-O-tridecyl ester of2-deoxy-D-glucose, 3-O-pentadecyl ester of 2-deoxy-D-glucose, 4-O-propylester of 2-deoxy-D-glucose, 4-O-tridecyl ester of 2-deoxy-D-glucose,4-O-pentadecyl ester of 2-deoxy-D-glucose, 6-O-propyl ester of2-deoxy-D-glucose, 6-O-tridecyl ester of 2-deoxy-D-glucose,6-O-pentadecyl ester of 2-deoxy-D-glucose, and 5-thio-D-glucose, andmixtures thereof.

In some aspects, provided herein are methods for treating cancercomprising the step of administering to a subject diagnosed as havingthe tumor, using, for example, the diagnostic and prognostic methods andsystems described herein, at least one proteosome inhibitor in apharmaceutically acceptable carrier. In other aspects, provided hereinare methods for treating a tumor comprising the step of administering toa subject diagnosed as having the tumor at least one histone deactylaseinhibitor in a pharmaceutically acceptable carrier. In another aspect,provided herein is a method for treating a tumor comprising the step ofadministering to a subject diagnosed as having the tumor, using, forexample, the diagnostic and prognostic methods and systems describedherein, at least one glycolytic inhibitor in a pharmaceuticallyacceptable carrier. In some embodiments of these aspects and all suchaspects described herein, the diagnosis of the tumor in the subject wasperformed according to determination of the TGS signature using themethods, systems, and kits described herein.

In other aspects, provided herein are methods for treating a basal-likebreast tumor comprising the step of administering to a subject diagnosedas having the basal-like breast tumor, using, for example, thediagnostic and prognostic methods and systems described herein, at leastone proteosome inhibitor in a pharmaceutically acceptable carrier. Insome aspects, provided herein are methods for treating a basal-likebreast tumor comprising the step of administering to a subject diagnosedas having the basal-like breast tumor, using, for example, thediagnostic and prognostic methods and systems described herein, at leastone histone deactylase inhibitor in a pharmaceutically acceptablecarrier. In other aspects, provided herein are method for treating abasal-like breast tumor comprising the step of administering to asubject diagnosed as having the basal-like breast tumor, using, forexample, the diagnostic and prognostic methods and systems describedherein, at least one glycolytic inhibitor in a pharmaceuticallyacceptable carrier.

The “agents” and “inhibitors” used in the therapeutic methods describedherein can be selected from a group of a chemical, small molecule,chemical entity, nucleic acid sequences, nucleic acid analogues orprotein or polypeptide or analogue of fragment thereof specific for oneor more a malignancy associated response signature biomarkers or genesencoding a malignancy associated response signature biomarker. Nucleicacid sequences include, for example, but not limited to, nucleic acidsequence encoding proteins that act as repressors in the various targetpathways described herein, antisense molecules, ribozymes, smallinhibitory nucleic acid sequences, for example but not limited to RNAi,shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.,specific for or directed against one or more genes encoding a malignancyassociated response signature biomarker, such as for example, one ormore of SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72,77, 78, 84, 91, 93; one or more of SEQ ID NOs: 1, 2, 4, 7, 29, 33, 34,and 38; or SEQ ID NO: 134, for example. One of skill in the art, knowingthe sequence a desired malignancy associated response signaturebiomarker to target, can design suitable inhibitory nucleic acidsequences using techniques known in the art or obtain ones from acommercial vendor, as described herein.

A protein and/or peptide agent or fragment thereof, can be any proteinof interest, for example, but not limited to; mutated proteins;therapeutic proteins; truncated proteins, wherein the protein isnormally absent or expressed at lower levels in the cell. Proteins ofinterest can be selected from a group comprising; mutated proteins,genetically engineered proteins, peptides, synthetic peptides,recombinant proteins, chimeric proteins, antibodies, humanized proteins,humanized antibodies, chimeric antibodies, modified proteins andfragments thereof. A therapeutic agent also includes any chemical,entity or moiety, including without limitation synthetic andnaturally-occurring non-proteinaceous entities. In certain embodiments,the agent is a small molecule having a chemical moiety. Such chemicalmoieties can include, for example, unsubstituted or substituted alkyl,aromatic, or heterocyclyl moieties and typically include at least anamine, carbonyl, hydroxyl or carboxyl group, frequently at least two ofthe functional chemical groups, including macrolides, leptomycins andrelated natural products or analogues thereof. Therapeutic agents can beknown to have a desired activity and/or property, or can be selectedfrom a library of diverse compounds.

As used herein, the term “small molecule” refers to a chemical agentwhich can include, but is not limited to, a peptide, a peptidomimetic,an amino acid, an amino acid analog, a polynucleotide, a polynucleotideanalog, an aptamer, a nucleotide, a nucleotide analog, an organic orinorganic compound (e.g., including heterorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

The term “inhibitor” refers to an agent that inhibits expression of apolypeptide or polynucleotide, or binds to, partially or totally blocksstimulation, decreases, prevents, delays activation, inactivates,desensitizes, or down regulates the activity of the polypeptide or thepolynucleotide. Inhibitors are agents that, e.g., inhibit expression,e.g., translation, post-translational processing, stability,degradation, or nuclear or cytoplasmic localization of a polypeptide, ortranscription, post transcriptional processing, stability or degradationof a polynucleotide or bind to, partially or totally block stimulation,DNA binding, transcription factor activity or enzymatic activity,decrease, prevent, delay activation, inactivate, desensitize, or downregulate the activity of a polypeptide or polynucleotide. An inhibitorcan act directly or indirectly. Inhibition is achieved when the activityvalue of a polypeptide or polynucleotide is about at least 10% less, atleast 20% less, at least 30% less, at least 40% less, at least 50% less,at least 60% less, at least 70% less, at least 80% less, at least 90%less, or absent or undetectable in comparison to a reference or controllevel in the absence of the inhibitor.

Accordingly, in some embodiments, an inhibitor for use in thetherapeutic methods described herein is an siRNA agent, an antisense RNAagent, a small molecule agent, an antibody or antigen-binding fragmentthereof specific for or directed against one or more malignancyassociated response signature biomarkers of Table 3 (SEQ ID NOs: 1-154).In some such embodiments, the malignancy associated response signaturebiomarker is a proteasomal pathway biomarker, a histone descetylasepathway biomarker, a glycolysis pathway biomarker, or any combinationthereof. In some embodiments, an inhibitor for use in the therapeuticmethods described herein is an siRNA agent, an antisense RNA agent, asmall molecule agent, an antibody or antigen-binding fragment thereofspecific for or directed against one or more of SEQ ID NOs: 1-4, 6, 7,13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93. In someembodiments, an inhibitor for use in the therapeutic methods describedherein is an siRNA agent, an antisense RNA agent, a small moleculeagent, an antibody or antigen-binding fragment thereof specific for ordirected against one or more of SEQ ID NOs: 1, 2, 4, 7, 29, 33, 34, and38. In some embodiments, an inhibitor for use in the therapeutic methodsdescribed herein is an siRNA agent, an antisense RNA agent, a smallmolecule agent, an antibody or antigen-binding fragment thereof specificfor or directed against SEQ ID NO: 134,

Therapeutic compositions comprising inhibitors, such as small molecules,drugs, siRNA or antisense RNA agents, of malignancy associated responsesignature biomarkers useful for practicing the therapeutic methodsdescribed herein comprise a physiologically tolerable carrier togetherwith an active compound, such as a proteosome inhibitor, histonedeacetylase inhibitor, or glycolytic inhibitor, as described herein,dissolved or dispersed therein as an active ingredient. In a preferredembodiment, the therapeutic composition is not immunogenic whenadministered to a mammal or human patient for therapeutic purposes,unless so desired. As used herein, the terms “pharmaceuticallyacceptable”, “physiologically tolerable” and grammatical variationsthereof, as they refer to compositions, carriers, diluents and reagents,are used interchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirableor unacceptable physiological effects such as toxicity, nausea,dizziness, gastric upset, immune reaction and the like. Apharmaceutically acceptable carrier will not promote the raising of animmune response to an agent with which it is admixed, unless so desired.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance theeffectiveness of the active ingredient. Physiologically tolerablecarriers are well known in the art. Exemplary liquid carriers aresterile aqueous solutions that contain no materials in addition to theactive ingredients and water, or contain a buffer such as sodiumphosphate at physiological pH value, physiological saline or both, suchas phosphate-buffered saline. Saline-based carriers are most useful forthe administration of cells or cell preparations. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes.

Dosage and administration of the therapeutic compositions comprising,for example, inhibitor agents that specifically inhibit one or more ofSEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78,84, 91, 93, described herein vary with the subject to be treated and thetherapeutic approach taken in a given instance. The success of atreatment can be evaluated by the ordinarily skilled clinician bymonitoring one or more symptoms or markers of the breast cancer beingtreated. Effective treatment includes any statistically significantimprovement in one or more indicia of the disease. Where appropriate, aclinically accepted grade or scaling system for the given disease ordisorder can be applied, with an improvement in the scale or grade beingindicative of effective treatment. Depending upon the therapeuticcomposition, various subject parameters, and the mode of delivery,effective dosages can include, for example, 1 ng/kg of body weight up toa gram or more per kg of body weight and any amount in between.Preferred amounts can be, for example, in the range of 5 μg/kg bodyweight to 500 mg/kg of body weight or any amount in between. Dosages insuch ranges can be administered once, twice, three times, four times ormore per day, or every two days, every three days, every four days, oncea week, twice a month, once a month or less frequently over a durationof days, weeks or months, depending on the breast cancer being treated,and as determined by a clinician. Sustained release formulations of thetherapeutic compositions targeting one or more TGS biomarkers arespecifically contemplated herein. Continuous, relatively low doses arealso contemplated after an initial higher therapeutic dose.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. Exemplary modes of administrationof the therapeutic compositions described herein, include, but are notlimited to, injection, infusion, inhalation (e.g., intranasal orintratracheal), ingestion, rectal, and topical (including buccal andsublingual) administration. The phrases “parenteral administration” and“administered parenterally” as used herein, refer to modes ofadministration other than enteral and topical administration, usually byinjection. As used herein, “injection” includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein refer toadministration of a therapeutic composition other than directly into atarget site, tissue, or organ, such as the lung, such that it enters thesubject's circulatory system and, thus, is subject to metabolism andother like processes.

Systems

Also provided herein, in other aspects and embodiments are systems (andcomputer readable media for causing computer systems) to perform methodsfor classifying, determining or diagnosing the type of a cancer ortumor, such as a breast tumor from a patient, or providing a prognosisto a cancer patient.

Accordingly, in some aspects, provided herein are systems forclassifying, determining or diagnosing the type of a cancer or tumor,such as a breast tumor from a patient, or providing a prognosis to atumor patient. Such systems comprise: (a) a determination moduleconfigured to receive a biological sample, measure expression levels inthe biological sample of at least 2 of the 154 biomarkers of the triplenegative breast cancer gene signature biomarker genes provided in Table3 (SEQ ID NOs: 1-154), and to output information of the expressionlevels of the at least 2 of the 154 biomarkers of the triple negativebreast cancer gene signature biomarker genes in the biological sample;(b) a storage device configured to store the output information of theexpression levels of the at least 2 of the 154 biomarkers of the triplenegative breast cancer gene signature biomarker genes from thedetermination module; (c) a comparison module adapted to receive inputfrom the storage device and compare the data stored on the storagedevice with reference expression level data of each of the at least 2 ofthe 154 biomarkers of the triple negative breast cancer gene signaturebiomarker genes, wherein if the expression level data of the at least 2of the 154 biomarkers of the triple negative breast cancer genesignature biomarker genes in the biological sample is increased relativeto the reference expression level data, the comparison module providesinformation to an output module that the biological sample is associatedwith a subject that has an epithelial progenitor cell tumor, a poorprognosis tumor or a highly malignant tumor, or a subject having a poorprognosis of breast cancer; and (d) an output module for displaying theinformation to the user.

Embodiments of the systems provided herein can be described throughfunctional modules, which are defined by computer executableinstructions recorded on computer readable media and which cause acomputer to perform method steps when executed. The modules describedherein are segregated by function for the sake of clarity. However, itshould be understood that the modules/systems need not correspond todiscreet blocks of code and the described functions can be carried outby the execution of various code portions stored on various media andexecuted at various times. Furthermore, it should be appreciated thatthe modules can perform other functions, thus the modules are notlimited to having any particular functions or set of functions.

The computer readable storage media can be any available tangible mediathat can be accessed by a computer. Computer readable storage mediaincludes volatile and nonvolatile, removable and non-removable tangiblemedia implemented in any method or technology for storage of informationsuch as computer readable instructions, data structures, program modulesor other data. Computer readable storage media includes, but is notlimited to, RAM (random access memory), ROM (read only memory), EPROM(erasable programmable read only memory), EEPROM (electrically erasableprogrammable read only memory), USB memory, flash memory or other memorytechnology, CD-ROM (compact disc read only memory), DVDs (digitalversatile disks) or other optical storage media, magnetic cassettes,magnetic tape, magnetic disk storage or other magnetic storage media,cloud server memory systems, other types of volatile and non-volatilememory, and any other tangible medium which can be used to store thedesired information and which can accessed by a computer including andany suitable combination of the foregoing.

Computer-readable data embodied on one or more computer-readable storagemedia can define instructions, for example, as part of one or moreprograms, that as a result of being executed by a computer, instruct thecomputer to perform one or more of the functions described herein,and/or various embodiments, variations and combinations thereof. Suchinstructions can be written in any of a plurality of programminglanguages, for example, Java, J#, Visual Basic, C, C#, C++, Fortran,Pascal, Eiffel, Basic, COBOL assembly language, and the like, or any ofa variety of combinations thereof. The computer-readable storage mediaon which such instructions are embodied can reside on one or more of thecomponents of either of a system, or a computer readable storage mediumdescribed herein, or can be distributed across one or more of suchcomponents.

The computer-readable storage media can be transportable such that theinstructions stored thereon can be loaded onto any computer resource toimplement the aspects of the present invention discussed herein. Inaddition, it should be appreciated that the instructions stored on thecomputer-readable medium, described above, are not limited toinstructions embodied as part of an application program running on ahost computer. Rather, the instructions can be embodied as any type ofcomputer code (e.g., software or microcode) that can be employed toprogram a computer to implement aspects of the present invention. Thecomputer executable instructions can be written in a suitable computerlanguage or combination of several languages. Basic computationalbiology methods are known to those of ordinary skill in the art and aredescribed in, for example, Setubal and Meidanis et al., Introduction toComputational Biology Methods (PWS Publishing Company, Boston, 1997);Salzberg, Searles, Kasif, (Ed.), Computational Methods in MolecularBiology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler,Bioinformatics Basics: Application in Biological Science and Medicine(CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: APractical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc.,2nd ed., 2001).

The functional modules of certain embodiments of the systems describedherein include, at minimum, a determination module or device, a storagemodule or device, a comparison module or device, and an output module ordevice or display module or device. The functional modules can beexecuted on one, or multiple, computers, or by using one, or multiple,computer networks. The determination system has computer executableinstructions to provide e.g., expression information in computerreadable form.

The determination system can comprise any system for determining orassaying expression levels of two or more genes of SEQ ID NOs.: 1-154.Such systems can include, but are not limited to, PCR or quantitativePCR machines or devices, microarray devices or systems, Northern blotanalysis systems, ELISA etc., as known to one of ordinary skill in theart.

The information determined in the determination system can be read bythe storage device. As used herein the “storage device” is intended toinclude any suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples of anelectronic apparatus suitable for use with the present invention includea stand-alone computing apparatus, data telecommunications networks,including local area networks (LAN), wide area networks (WAN), Internet,Intranet, and Extranet, local and remote servers, and local anddistributed computer processing systems. Storage devices also include,but are not limited to: magnetic storage media, such as floppy discs,hard disc storage media, remote or local servers, magnetic tape, opticalstorage media such as CD-ROM, DVD, electronic storage media such as RAM,ROM, EPROM, EEPROM and the like, general hard disks and hybrids of thesecategories such as magnetic/optical storage media. The storage device isadapted or configured for having recorded thereon nucleic acid sequenceinformation. Such information can be provided in digital form that canbe transmitted and read electronically, e.g., via the Internet, ondiskette, via USB (universal serial bus) or via any other suitable modeof communication.

As used herein, “stored” refers to a process for encoding information onthe storage device. Those skilled in the art can readily adopt any ofthe presently known methods for recording information on known media togenerate manufactures comprising information relating to immunestimulating microbes.

In some embodiments of the aspects and embodiments described herein, thereference data stored in the storage device to be read by the comparisonmodule is e.g., expression data of two or more genes of Table 3 (SEQ IDNOs: 1-154) obtained from a patient sample, such as a patient having atriple-negative breast cancer.

The “comparison module” can use a variety of available software programsand formats for the comparison operative to compare expressioninformation data of the two or more TGS genes or biomarkers of Table3(SEQ ID NOs: 1-154) determined in the determination system to one ormore reference samples and/or stored reference data. In some embodimentsof the systems described herein, the comparison module is configured touse pattern recognition techniques to compare information from one ormore entries to one or more reference data patterns. The comparisonmodule can be configured using existing commercially-available orfreely-available software for comparing patterns, and can be optimizedfor particular data comparisons that are conducted. The comparisonmodule provides computer readable information related to the expressionlevels of two or more of the TGS genes of Table 3 (SEQ ID NOs: 1-154).

The comparison module, or any other module of the invention, can includean operating system (e.g., UNIX) on which runs a relational databasemanagement system, a World Wide Web application, and a World Wide Webserver. World Wide Web application includes the executable codenecessary for generation of database language statements (e.g.,Structured Query Language (SQL) statements). Generally, the executableswill include embedded SQL statements. In addition, the World Wide Webapplication can include a configuration file which contains pointers andaddresses to the various software entities that comprise the server aswell as the various external and internal databases which must beaccessed to service user requests. The Configuration file also directsrequests for server resources to the appropriate hardware—as may benecessary should the server be distributed over two or more separatecomputers. In one embodiment, the World Wide Web server supports aTCP/IP protocol. Local networks such as this are sometimes referred toas “Intranets.” An advantage of such Intranets is that they allow easycommunication with public domain databases residing on the World WideWeb (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in someembodiments, users can directly access data (via Hypertext links forexample) residing on Internet databases using a HTML interface providedby Web browsers and Web servers.

The comparison module provides a computer readable comparison resultthat can be processed in computer readable form by predefined criteria,or criteria defined by a user, to provide a content based in part on thecomparison result that may be stored and output as requested by a userusing a display module or output device.

The content based on the comparison result, can be, for example, thetype of tumor, or the prognosis of the tumor. Alternatively, oradditionally, the content based on the comparison result can be afurther treatment step indicated for the patient, e.g., administrationof a proteasome inhibitor or histone deacetylase inhibitor.

In some embodiments of the systems described herein, the content basedon the comparison result is displayed on a computer monitor. In someembodiments of the systems described herein, the content based on thecomparison result is displayed through printable media. The displaymodule can be any suitable device configured to receive from a computerand display computer readable information to a user. Non-limitingexamples include, for example, general-purpose computers such as thosebased on Intel PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC,Hewlett-Packard PA-RISC processors, any of a variety of processorsavailable from Advanced Micro Devices (AMD) of Sunnyvale, Calif., tabletor mobile phone devices, or any other type of processor, visual displaydevices such as flat panel displays, cathode ray tubes and the like, aswell as computer printers of various types.

In some embodiments of the systems described herein, a World Wide Webbrowser is used for providing a user interface for display of thecontent based on the comparison result. It should be understood thatother modules of the invention can be adapted to have a web browserinterface. Through the Web browser, a user may construct requests forretrieving data from the comparison module. Thus, the user willtypically point and click to user interface elements such as buttons,pull down menus, scroll bars and the like conventionally employed ingraphical user interfaces.

The modules of the machine, or those used in the computer readablemedium, can assume numerous configurations. For example, function may beprovided on a single machine or distributed over multiple machines.

Accordingly, the methods described herein therefore provide for systems(and computer readable media for causing computer systems) to performmethods for determining or diagnosing the type of a cancer or tumor in apatient, such as a breast tumor from a patient, or providing a prognosisto a tumor patient.

Systems and computer readable media described herein are merelyillustrative embodiments of the invention for performing methods ofdiagnosis in an individual, and are not intended to limit the scope ofthe invention. Variations of the systems and computer readable mediadescribed herein are possible and are intended to fall within the scopeof the invention.

Kits for Detection of Malignancy Associated Response SignatureBiomarkers

Conveniently, in some aspects, expression of malignancy associatedresponse signature biomarkers in the methods described herein can beevaluated using a kit comprising at least one malignancy associatedresponse signature biomarker probe suitable for detecting one or moremalignancy associated response signature biomarkers. As used herein, a“malignancy associated response signature probe” can include anymolecule capable of detecting a malignancy associated response signaturebiomarker including, but not limited to, monoclonal and polyclonalantibodies and fragments thereof, and oligonucleotides. For example, thekit can comprise an antibody specific for an epitope of a malignancyassociated response signature biomarker protein encoded by an malignancyassociated response signature biomarker gene, an oligonucleotide probecomplementary to at least a portion of a malignancy associated responsesignature biomarker gene or to at least a portion an RNA (e.g., mRNA)encoded by a malignancy associated response signature biomarker gene, orprimer pairs suitable for evaluating gene expression of a malignancyassociated response signature biomarker by a polymerase chain reaction(PCR)-based method, such as real time PCR or reverse transcription PCR.Other methodologies for measuring expression of a malignancy associatedresponse signature biomarker include ribonuclease protection assays, 51nuclease assays, and Northern blot analyses. Optionally, the kitsinclude instructions for detecting malignancy associated responsesignature biomarker detection or for performing the methods or diagnosisand prognosis described herein

In some embodiments, the kit can comprise a microarray that can be usedto determine expression of at least one malignancy associated responsesignature biomarker in a tumor sample and instructions for analyzing theinformation for use in the methods described herein. The microarrayincludes at least one oligonucleotide comprising a sequence of at leastone of the malignancy associated response signature biomarker from thegroup of markers listed in Table 3 (SEQ ID NOs: 1-154). More preferably,the microarray includes oligonucleotides comprising a sequence of atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 23, at least 25, at least 30, at least35, at least 40, at least 45, at least 50, at least 100, at least 150,or all 154 of the 154 malignancy associated response signaturebiomarkers described in Table 3 (SEQ ID NOs: 1-154). The term“microarray,” as used herein, refers to an ordered arrangement ofhybridizable array elements, e.g. oligonucleotide probes, on asubstrate, e.g. glass slide or silica. Suitably, the microarraycomprises control probes to allow for detection of expression levelsthat can be used to determine enrichment of malignancy associatedresponse signature biomarker genes relative to a control or referencesample.

Screening Methods

The malignancy associated response signature biomarkers described hereinare also useful in screening and identifying novel therapeutic agentsagainst malignant and poor prognosis breast cancers, such as basal-likebreast tumors.

Accordingly, in one aspect, provided herein is a method of identifying acandidate therapeutic agent against a tumor, such as a basal-like breasttumor, the method comprising the steps of (a) exposing a BPLER cellculture to a test agent, wherein said BPLER cell culture comprises humanbreast primary epithelial cells (BPE) transformed with a defined set ofgenetic elements (BPLER cells); (b) measuring the expression of at least10 of the 154 of the genes of a malignancy associated response signaturein the culture, wherein the malignancy associated response signaturecomprises an expression pattern of a set of 10-154 biomarkers set forthin Table 3 (SEQ ID NOs: 1-154); (c) comparing the expression of the sameat least 10 genes of malignancy associated response signature as wasmeasured in step (b) to an expression signature reference from a BPLERcell culture that has not been exposed to the test agent, wherein adecrease in expression of at least 5 of the at least 10 genes in thetest culture compared to the expression signature of the referenceculture indicates that the test agent is a candidate therapeutic agentagainst the tumor, e.g., basal-like breast tumor.

The term “screening” as used herein refers to the use of cells andtissues in the laboratory to identify agents with a specific function,e.g., inhibiting activity against a malignancy associated responsesignature biomarker. “High-throughput screening technologies” refer toplatforms and assays used to rapidly test thousands of test compounds.For example, reporter systems used in cell lines can be used to assesswhether compounds activate or inhibit particular signaling pathways ofinterest, such as the ubiquitin-proteasome pathway.

The “candidate agent” used in the screening methods described herein canbe selected from a group of a chemical, small molecule, chemical entity,nucleic acid sequences, an action; nucleic acid analogues or protein orpolypeptide or analogue of fragment thereof. Nucleic acid sequencesinclude, for example, but not limited to, nucleic acid sequence encodingproteins that act as transcriptional repressors, antisense molecules,ribozymes, small inhibitory nucleic acid sequences, for example but notlimited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisenseoligonucleotides etc. A protein and/or peptide agent or fragmentthereof, can be any protein of interest, for example, but not limitedto; mutated proteins; therapeutic proteins; truncated proteins, whereinthe protein is normally absent or expressed at lower levels in the cell.Proteins of interest can be selected from a group comprising; mutatedproteins, genetically engineered proteins, peptides, synthetic peptides,recombinant proteins, chimeric proteins, antibodies, humanized proteins,humanized antibodies, chimeric antibodies, modified proteins andfragments thereof. A candidate agent also includes any chemical, entityor moiety, including without limitation synthetic andnaturally-occurring non-proteinaceous entities. In certain embodiments,the candidate agent is a small molecule having a chemical moiety. Suchchemical moieties can include, for example, unsubstituted or substitutedalkyl, aromatic, or heterocyclyl moieties and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, frequently at least twoof the functional chemical groups, including macrolides, leptomycins andrelated natural products or analogues thereof. Candidate agents can beknown to have a desired activity and/or property, or can be selectedfrom a library of diverse compounds.

As used herein, the term “small molecule” refers to a chemical agentwhich can include, but is not limited to, a peptide, a peptidomimetic,an amino acid, an amino acid analog, a polynucleotide, a polynucleotideanalog, an aptamer, a nucleotide, a nucleotide analog, an organic orinorganic compound (e.g., including heterorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

The term “inhibitor” refers to an agent that inhibits expression of apolypeptide or polynucleotide, or binds to, partially or totally blocksstimulation, decreases, prevents, delays activation, inactivates,desensitizes, or down regulates the activity of the polypeptide or thepolynucleotide. Inhibitors are agents that, e.g., inhibit expression,e.g., translation, post-translational processing, stability,degradation, or nuclear or cytoplasmic localization of a polypeptide, ortranscription, post transcriptional processing, stability or degradationof a polynucleotide or bind to, partially or totally block stimulation,DNA binding, transcription factor activity or enzymatic activity,decrease, prevent, delay activation, inactivate, desensitize, or downregulate the activity of a polypeptide or polynucleotide. An inhibitorcan act directly or indirectly. Inhibition is achieved when the activityvalue of a polypeptide or polynucleotide is about at least 10% less, atleast 20% less, at least 30% less, at least 40% less, at least 50% less,at least 60% less, at least 70% less, at least 80% less, at least 90%less, or absent or undetectable in comparison to a reference or controllevel in the absence of the inhibitor.

Also included as candidate agents are pharmacologically active drugs,genetically active molecules, etc. Such candidate agents of interestinclude, for example, chemotherapeutic agents, hormones or hormoneantagonists, growth factors or recombinant growth factors and fragmentsand variants thereof. Exemplary of pharmaceutical agents suitable foruse with the screening methods described herein are those described in,“The Pharmacological Basis of Therapeutics,” Goodman and Gilman,McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections:Water, Salts and Ions; Drugs Affecting Renal Function and ElectrolyteMetabolism; Drugs Affecting Gastrointestinal Function; Chemotherapy ofMicrobial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Acting onBlood-Forming organs; Hormones and Hormone Antagonists; Vitamins,Dermatology; and Toxicology, all of which are incorporated herein byreference in their entireties. Also included are toxins, and biologicaland chemical warfare agents, for example see Somani, S. M. (Ed.),“Chemical Warfare Agents,” Academic Press, New York, 1992), the contentsof which is herein incorporated in its entirety by reference.

Candidate agents, such as chemical compounds, can be obtained from awide variety of sources including libraries of synthetic or naturalcompounds. For example, numerous means are available for random anddirected synthesis of a wide variety of organic compounds, includingbiomolecules, including expression of randomized oligonucleotides andoligopeptides. Alternatively, libraries of natural compounds in the formof bacterial, fungal, plant and animal extracts are available or readilyproduced. Additionally, natural or synthetically produced libraries andcompounds are readily modified through conventional chemical, physicaland biochemical means, and may be used to produce combinatoriallibraries. Known pharmacological agents can be subjected to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the candidatecompounds for use in the screening methods described herein are known inthe art and include, for example, those such as described in R. Larock(1989) Comprehensive Organic Transformations, VCH Publishers; T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nded., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof, the contents ofeach of which are herein incorporated in their entireties by reference.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233, the contents of each ofwhich are herein incorporated in their entireties by reference.

Libraries of candidate agents can be presented in solution (e.g.,Houghten (1992), Biotechniques 13:412-421), or on beads (Lam (1991),Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No.5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladnersupra.), the contents of each of which are herein incorporated in theirentireties by reference.

High through-put screening is a process in which libraries of compoundsare tested for a given activity. High through-put screening seeks toscreen large numbers of compounds rapidly and in parallel. For example,using microtiter plates, robotic equipment, and automated assayequipment, a pharmaceutical company may perform as many as 100,000assays per day in parallel.

The compound screening assays described herein can involve more than onemeasurement of an observable reporter function, expression or activityof a malignancy associated response biomarker gene. Multiplemeasurements allow for following the biological activity over incubationtime with the test compound. In one embodiment, the expression oractivity of a malignancy associated response biomarker gene is measuredat a plurality of times to allow monitoring of the effects of the testcompound at different incubation times.

Accordingly, in some embodiments of the screening aspects, providedherein is a high throughput automated screening system for identifying acandidate therapeutic agent against a tumor, such as a basal-like breasttumor, the system comprising:

a) a high throughput candidate agent screening culture, connected to

b) a computer processor and a computer-readable physical medium withsoftware instructions encoded thereupon for a process, executable bysaid processor, said instructions comprising:

(i) instructions for receiving data regarding the measurement of theexpression of at least 10 of the 154 of the genes of a malignancyassociated response in a high-throughput screening culture, wherein themalignancy associated response comprises an expression pattern of a setof 10-154 biomarkers set forth in Table 3 (SEQ ID NOs: 1-154); (ii)instructions for comparing the expression data received in (i) withreference signature expression data of the same at least 10 genes ofmalignancy associated response from a cell culture that has not beenexposed to the test agents, wherein said comparing identifies whetherthere is a decrease in expression of at least 5 of the at least 10 genesin the test culture compared to the expression signature of thereference culture and thus indicates that the test agent is a candidatetherapeutic agent against the tumor, such as basal-like breast tumor;and (iii) outputting the result of said comparison to a user interface.

The high-throughput screening and other system methods described hereinbe implemented using any device capable of implementing the methods.Examples of devices that can be used include but are not limited toelectronic computational devices, including computers of all types. Whenthe methods described herein are implemented in a computer, the computerprogram that can be used to configure the computer to carry out thesteps of the methods can be contained in any computer readable mediumcapable of containing the computer program. Examples of computerreadable medium that can be used include but are not limited todiskettes, CD-ROMs, DVDs, ROM, RAM, USB devices, and other memory andcomputer storage devices. The computer program that can be used toconfigure the computer to carry out the steps of the methods can also beprovided over an electronic network, for example, over the internet,world-wide web, an intranet, or other network.

In one such example, the methods described herein can be implemented ina system comprising a processor and a computer readable medium thatincludes program code means for causing the system to carry out thesteps of the methods described in the present invention. The processorcan be any processor capable of carrying out the operations needed forimplementation of the methods. The program code means can be any codethat when implemented in the system can cause the system to carry outthe steps of the methods described in the present invention. Examples ofprogram code means include but are not limited to instructions to carryout the methods described in this patent written in a high levelcomputer language such as C++, Java, or Fortran; instructions to carryout the methods described herein written in a low level computerlanguage such as assembly language; or instructions to carry out themethods described herein in a computer executable form such as compiledand linked machine language.

Some aspects and embodiments disclosed herein can be illustrated by, forexample any of the following numbered paragraphs:

-   1. A malignancy associated response signature for a    cancer-initiating cell consisting essentially of an expression    pattern of a set of biomarkers set forth in SEQ ID NOs: 1-4, 6, 7,    13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151,    wherein at least 5 of the 23 biomarkers have increased expression    compared to a reference value.-   2. The malignancy associated response signature of paragraph 1,    wherein the cancer-initiating cell is a breast cancer-initiating    cell.-   3. The malignancy associated response signature of paragraph 1,    wherein the cancer-initiating cell is a triple-negative breast    cancer-initiating cell.-   4. The malignancy associated response signature of paragraph 1,    wherein the cancer-initiating cell is a Luminal B breast    cancer-initiating cell-   5. The malignancy associated response signature of paragraph 1,    wherein the cancer-initiating cell is an epithelial breast    cancer-initiating cell.-   6. The malignancy associated response signature of any one of    paragraphs 1-5, wherein the expression of the at least 5 of the 23    biomarkers is increased at least 1.8-fold compared to the reference    value.-   7. A malignancy associated response signature for a    cancer-initiating cell comprising an expression pattern of a set of    at least 10 biomarkers set forth in SEQ ID NOs: 1-154, wherein at    least 10 of the 154 markers have increased expression compared to a    reference value.-   8. The malignancy associated response signature of paragraph 7,    wherein the cancer-initiating cell is a breast cancer-initiating    cell.-   9. The malignancy associated response signature of paragraph 7,    wherein the cancer-initiating cell is a triple-negative breast    cancer-initiating cell.-   10. The malignancy associated response signature of paragraph 7,    wherein the cancer-initiating cell is a Luminal B breast    cancer-initiating cell.-   11. The malignancy associated response signature of paragraph 7,    wherein the cancer-initiating cell is an epithelial breast    cancer-initiating cell.-   12. The malignancy associated response signature of any one of    paragraphs 7-11, wherein the expression of the at least 5 biomarkers    is increased at least 1.8-fold compared to the reference value.-   13. The malignancy associated response signature of any one of    paragraphs 7-12, wherein at least 2 of the set of at least 10    biomarkers set forth in SEQ ID NOs: 1-154 is selected from the group    consisting of SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38,    66, 72, 77, 78, 84, 91, 93, and 151.-   14. A malignancy associated response signature for a    cancer-initiating cell susceptible to proteasomal gene inhibition    comprising an expression pattern of a set of at least 3 biomarkers    set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60,    74, 76, and 149, wherein at least 3 of the 15 biomarkers have    increased expression compared to a reference value.-   15. A malignancy associated response signature for a    cancer-initiating cell susceptible to mitosis gene inhibition    comprising an expression pattern of a set of at least 3 biomarkers    set forth in SEQ ID NOs: 9, 17, 22, 23, 31, 37, 35, 52, 68, 80, 101,    124, 130, and 137, wherein at least 3 of the 14 biomarkers have    increased expression compared to a reference value.-   16. A malignancy associated response signature for a    cancer-initiating cell susceptible to RNA splicing gene inhibition    comprising an expression pattern of a set of at least 3 biomarkers    set forth in SEQ ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57, 61, 123,    127, and 151, wherein at least 3 of the 13 biomarkers have increased    expression compared to a reference value.-   17. A malignancy associated response signature for a    cancer-initiating cell susceptible to molecular transport gene    inhibition comprising an expression pattern of a set of at least 3    biomarkers set forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56, 64, 91,    96, 98, 108, 121, and 147, wherein at least 3 of the 14 biomarkers    have increased expression compared to a reference value.-   18. A malignancy associated response signature for a    cancer-initiating cell susceptible to metabolic gene inhibition    comprising an expression pattern of a set of at least 3 biomarkers    of SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141,    and 153, wherein at least 3 of the 13 biomarkers have increased    expression compared to a reference value.-   19. The malignancy associated response signature of any one of    paragraphs 14-18, wherein the cancer-initiating cell is a breast    cancer-initiating cell.-   20. The malignancy associated response signature of any one of    paragraphs 14-18, wherein the cancer-initiating cell is a    triple-negative breast cancer-initiating cell.-   21. The malignancy associated response signature of any one of    paragraphs 14-18, wherein the cancer-initiating cell is a Luminal B    breast cancer-initiating cell.-   22. The malignancy associated response signature of any one of    paragraphs 14-18, wherein the cancer-initiating cell is an    epithelial breast cancer-initiating cell.-   23. The malignancy associated response signature of any one of    paragraphs 14-22, wherein the expression of the at least 3    biomarkers is at least 1.8-fold increased compared to the reference    value.-   24. A method of classifying a cancer in a subject in need thereof,    the method comprising:    -   a. assaying expression of ten or more of the 154 malignancy        associated response signature biomarkers of SEQ ID NOs: 1-154 in        a biological sample obtained from the subject having a cancer,        and    -   b. comparing the expression of the ten or more of the 154        malignancy associated response signature biomarkers of SEQ ID        NOs: 1-154 in the biological sample obtained from the subject        having a cancer with a reference value, wherein increased        expression of 1.8-fold or greater of at least ten of the        biomarkers in the biological sample obtained from the subject        relative to the reference value indicates that the cancer is        classified as having a poor prognosis or being a malignant        cancer, and absence of increased expression of 1.8-fold or        greater of at least ten of the biomarkers relative to the        reference value indicates that the cancer does not have poor        prognosis or is not a malignant cancer.-   25. The method of paragraph 24, wherein the cancer is a breast    cancer.-   26. The method of paragraph 24, wherein the cancer is a    triple-negative breast cancer.-   27. The method of paragraph 24, wherein the cancer is a Luminal B    breast cancer.-   28. The method of paragraph 24, wherein the cancer is an epithelial    breast cancer.-   29. The method of any one of paragraphs 27-31, wherein if the cancer    is classified as having a poor prognosis or being a malignant    cancer, the method further comprises the step of administering at    least one proteasome inhibitor to the subject.-   30. The method of any one of paragraphs 24-29, wherein if the cancer    is classified as having a poor prognosis or being a malignant cancer    and wherein if at least two of the ten or more genes having    increased expression is a proteasomal malignancy associated response    signature biomarker of SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33,    34, 38, 60, 74, 76, and 149, the method further comprises the step    of administering at least one proteasome inhibitor to the subject.-   31. The method of any one of paragraphs 29 or 30, wherein the at    least one proteasome inhibitor specifically inhibits any of the    proteasomal malignancy associated response signature biomarkers set    forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74,    76, and 149.-   32. The method of any one of paragraphs 29-31, wherein the at least    one proteasome inhibitor specifically inhibits the malignancy    associated response signature biomarker set forth in SEQ ID NOs: 134    (MCL-1).-   33. The method of any one of paragraphs 29-32, wherein the at least    one proteasome inhibitor is an siRNA or antisense RNA agent.-   34. The method of any one of paragraphs 29-30, wherein the at least    one proteasome inhibitor is bortezomib.-   35. The method of any one of paragraphs 24-34, wherein if the cancer    is classified as having a poor prognosis or being a malignant    cancer, the method further comprises the step of administering at    least one histone deacetylase inhibitor to the subject.-   36. The method of paragraph 35, wherein the at least one histone    deacetylase inhibitor is an siRNA or antisense RNA agent.-   37. The method of paragraph 35, wherein the at least one histone    deacetylase inhibitor is trichostatin A (TSA) or Vorinostat.-   38. The method of any one of paragraphs 24-37, wherein if the cancer    is classified as having a poor prognosis or being a malignant    cancer, the method further comprises the step of administering at    least one glygolytic inhibitor to the subject.-   39. The method of paragraph 38, wherein the at least one glycolysis    inhibitor is an siRNA or antisense RNA agent.-   40. The method of any one of paragraphs 24-39, wherein if the cancer    is classified as having a poor prognosis or being a malignant    cancer, relative survival rate, relative risk of metastasis,    treatment option, or any combination thereof is determined for the    subject.-   41. A method of classifying a cancer in a subject in need thereof,    the method comprising:    -   a. assaying expression of at least five biomarkers set forth in        SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72,        77, 78, 84, 91, 93, and 151 in a biological sample obtained from        the subject having a cancer, and    -   b. comparing the expression of the five or more of the 23        malignancy associated response signature biomarkers in the        biological sample obtained from the subject having a cancer with        a reference value, wherein increased expression of 1.8-fold or        greater of at least ten of the biomarkers in the biological        sample obtained from the subject relative to the reference value        indicates that the cancer is classified as having a poor        prognosis or being a malignant cancer, and absence of increased        expression of 1.8-fold or greater of at least ten of the        biomarkers relative to the reference value indicates that the        cancer does not have poor prognosis or is not a malignant        cancer.-   42. The method of paragraph 41, wherein the cancer is a breast    cancer.-   43. The method of paragraph 41, wherein the cancer is a    triple-negative breast cancer.-   44. The method of paragraph 41, wherein the cancer is a Luminal B    breast cancer.-   45. The method of paragraph 41, wherein the cancer is an epithelial    breast cancer.-   46. The method of any one of paragraphs 41-45, wherein if the cancer    is classified as having a poor prognosis or being a malignant    cancer, the method further comprises the step of administering at    least one proteasome inhibitor to the subject.-   47. The method of any one of paragraphs 41-45, wherein if the cancer    is classified as having a poor prognosis or being a malignant cancer    and wherein at least one of the five or more genes having increased    expression is a proteasomal malignancy associated response signature    biomarker of SEQ ID NOs: 1, 2, 4, 7, 29, 33, 34, and 38, the method    further comprises the step of administering at least one proteasome    inhibitor to the subject.-   48. The method of any one of paragraphs 46-47, wherein the at least    one proteasome inhibitor specifically inhibits any of the    proteasomal malignancy associated response signature biomarkers set    forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74,    76, and 149.-   49. The method of any one of paragraphs 46-47, wherein the at least    one proteasome inhibitor specifically inhibits the malignancy    associated response signature biomarker set forth in SEQ ID NOs: 134    (MCL-1).-   50. The method of any one of paragraphs 46-49, wherein the at least    one proteasome inhibitor is an siRNA or antisense RNA agent.-   51. The method of any one of paragraphs 46-47, wherein the at least    one proteasome inhibitor is bortezomib.-   52. A method of classifying a cancer in a subject in need thereof,    the method comprising:    -   a. assaying expression of at least three malignancy associated        response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4,        7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149 in a        biological sample obtained from the subject having a cancer, and    -   b. comparing the expression of the at least three malignancy        associated response signature biomarkers in the biological        sample obtained from the subject having a cancer with a        reference value, wherein increased expression of 1.8-fold or        greater of at least three of the biomarkers in the biological        sample obtained from the subject relative to the reference value        indicates that the cancer is classified as having a poor        prognosis or being a malignant cancer, and absence of increased        expression of 1.8-fold or greater of at least three of the        biomarkers relative to the reference value indicates that the        cancer does not have poor prognosis or is not a malignant        cancer.-   53. The method of paragraph 52, wherein the cancer is a breast    cancer.-   54. The method of paragraph 52, wherein the cancer is a    triple-negative breast cancer.-   55. The method of paragraph 52, wherein the cancer is a Luminal B    breast cancer.-   56. The method of paragraph 52, wherein the cancer is an epithelial    breast cancer.-   57. The method of any one of paragraphs 52-56, wherein if the cancer    is classified as having a poor prognosis or being a malignant    cancer, the method further comprises the step of administering at    least one proteasome inhibitor to the subject.-   58. The method of paragraph 57, wherein the at least one proteasome    inhibitor specifically inhibits any of the proteasomal malignancy    associated response signature biomarkers set forth in SEQ ID NOs: 1,    2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.-   59. The method of paragraph 57, wherein the at least one proteasome    inhibitor specifically inhibits the malignancy associated response    signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).-   60. The method of any one of paragraphs 57-59, wherein the at least    one proteasome inhibitor is an siRNA or antisense RNA agent.-   61. The method of paragraph 57, wherein the at least one proteasome    inhibitor is bortezomib.-   62. An assay comprising the steps of:    -   a. measuring expression of ten or more of the 154 malignancy        associated response signature biomarkers set forth in SEQ ID        NOs: 1-154 in a biological sample obtained from a subject having        cancer, and    -   b. comparing the expression of the ten or more of the 154        malignancy associated response signature biomarkers set forth in        SEQ ID NOs: 1-154 in the biological sample obtained from the        subject having a cancer with a reference value, wherein        increased expression of at least ten of the measured biomarkers        in the biological sample obtained from the subject relative to        the reference value diagnoses the patient as having a poor        prognosis or malignant cancer, and absence of increased        expression of at least ten of the measured biomarkers relative        to the reference value diagnoses the patient as not having a        poor prognosis or malignant cancer.-   63. The assay of paragraph 62, wherein the increased expression of    the ten or more of the 154 malignancy associated response signature    biomarkers in the biological sample determines a prognosis for the    subject having the cancer.-   64. The assay of paragraph 63, wherein the prognosis comprises    relative survival rate, relative risk of metastasis, treatment    option, or any combination thereof.-   65. The assay of any one of paragraphs 62-64, wherein the cancer is    a breast cancer.-   66. The assay of any one of paragraphs 62-64, wherein the cancer is    a triple-negative breast cancer.-   67. The assay of any one of paragraphs 62-64, wherein the cancer is    a Luminal B breast cancer.-   68. The assay of any one of paragraphs 62-64, wherein the cancer is    an epithelial breast cancer.-   69. The assay of any one of paragraphs 62-68, wherein if the subject    is diagnosed as having a poor prognosis or malignant cancer, the    subject is administered at least one proteasome inhibitor.-   70. The assay of any one of paragraphs 62-68, wherein if the subject    is diagnosed as having a poor prognosis or malignant cancer and    wherein at least two of the ten or more genes having increased    expression is a proteasomal malignancy associated response signature    biomarker of SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60,    74, 76, and 149, the subject is administered at least one proteasome    inhibitor.-   71. The assay of any one of paragraphs 69-70, wherein the at least    one proteasome inhibitor specifically inhibits any of the    proteasomal malignancy associated response signature biomarkers set    forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74,    76, and 149.-   72. The assay of any one of paragraphs 69-71, wherein the at least    one proteasome inhibitor specifically inhibits the malignancy    associated response signature biomarker set forth in SEQ ID NOs: 134    (MCL-1).-   73. The assay of any one of paragraphs 69-72, wherein the at least    one proteasome inhibitor is an siRNA or antisense RNA agent.-   74. The assay of any one of paragraphs 69-70, wherein the at least    one proteasome inhibitor is bortezomib.-   75. The assay of any one of paragraphs 69-74, wherein if the subject    is diagnosed as having a poor prognosis or malignant cancer, the    subject is administered at least one histone deacetylase inhibitor.-   76. The assay of paragraph 75, wherein the at least one histone    deacetylase inhibitor is an siRNA or antisense RNA agent.-   77. The assay of paragraph 75, wherein the at least one histone    deacetylase inhibitor is trichostatin A (TSA) or Vorinostat.-   78. The assay of any one of paragraphs 69-77, wherein if the subject    is diagnosed as having a poor prognosis or malignant cancer, the    subject is administered at least one glygolytic inhibitor.-   79. The assay of paragraph 78, wherein the at least one glycolysis    inhibitor is an siRNA or antisense RNA agent.-   80. An assay comprising the steps of:    -   a. dividing a cell culture grown from a biopsy obtained from a        subject having cancer into at least 5 separate cultures;    -   b. exposing each of the at least 5 separate cultures to a        different inhibitory agent, wherein each said different        inhibitory agent specifically inhibits a different malignancy        associated response signature biomarker set forth in SEQ ID NOs:        1-154    -   c. growing each of the at least 5 separate cell cultures of        step (b) for at least 12 hours;    -   d. measuring viability of the cells from each of the cultures of        step (c), wherein if the total viability of the cells in at        least 60% of the cultures is decreased by at least 25%, then the        biopsy obtained from the subject comprises cancer-initiating        cells.-   81. The assay of paragraph 80, wherein the inhibitory agents are    selected from siRNA agents, antisense RNA agents, or small    molecules, that specifically inhibit any of the cancer gene    signature biomarkers set forth in SEQ ID NOs: 1-154.-   82. The assay of any one of paragraphs 80-81, wherein the inhibitory    agents specifically inhibit any of the malignancy associated    response signature biomarkers set forth in SEQ ID NOs: 1-4, 6, 7,    13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151.-   83. The assay of any one of paragraphs 80-81, wherein at least one    inhibitory agent specifically inhibits any of the malignancy    associated response signature biomarkers set forth in SEQ ID NOs:    1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91,    93, and 151.-   84. The assay of any one of paragraphs 80-83, wherein at least one    inhibitory agent specifically inhibits any of the proteasomal    malignancy associated response signature biomarkers set forth in SEQ    ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.-   85. The assay of any one of paragraphs 80-84, wherein at least one    inhibitory agent specifically inhibits any of the mitosis cancer    gene signature biomarkers set forth in SEQ ID NOs: 9, 17, 22, 23,    31, 37, 35, 52, 68, 80, 101, 124, 130, and 137.-   86. The assay of any one of paragraphs 80-85, wherein at least one    inhibitory agent specifically inhibits any of the RNA splicing    malignancy associated response signature biomarkers set forth in SEQ    ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57, 61, 123, 127, and 151.-   87. The assay of any one of paragraphs 80-86, wherein at least one    inhibitory agent specifically inhibits any of the molecular    transport malignancy associated response signature biomarkers set    forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56, 64, 91, 96, 98, 108,    121, and 147.-   88. The assay of any one of paragraphs 80-87, wherein at least one    inhibitory agent specifically inhibits any of the metabolic    malignancy associated response signature biomarkers set forth in SEQ    ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and    153.-   89. A method for treating a cancer in a subject in need thereof    comprising the step of administering to a subject having a cancer    classified or diagnosed as having poor prognosis or being malignant,    using the method of any one of paragraphs 24-61, or the assay of any    one of paragraphs 62-79, at least one proteasome inhibitor in a    pharmaceutically acceptable carrier.-   90. The method of paragraph 89, wherein the at least one proteasome    inhibitor is an siRNA or antisense RNA agent.-   91. The method of any one of paragraphs 89-90, wherein the    proteasome inhibitor specifically inhibits a proteasomal malignancy    associated response signature biomarkers set forth in SEQ ID NOs: 1,    2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.-   92. The assay of any one of paragraphs 89-90, wherein the at least    one proteasome inhibitor specifically inhibits the malignancy    associated response signature biomarker set forth in SEQ ID NOs: 134    (MCL-1).-   93. The method of paragraph 89, wherein the proteasome inhibitor is    bortezomib.-   94. A method for treating a cancer in a subject in need thereof    comprising the step of administering to a subject having a cancer    classified or diagnosed as having poor prognosis or being malignant,    using the method of any one of paragraphs 24-61, or the assay of any    one of paragraphs 62-79, at least one histone deacetylase inhibitor    in a pharmaceutically acceptable carrier.-   95. The method of paragraph 92, wherein the at least one histone    deacetylase inhibitor is an siRNA or antisense RNA agent.-   96. The method of paragraph 93, wherein the at least one histone    deacetylase inhibitor is trichostatin A (TSA) or Vorinostat.-   97. A method for treating a cancer in a subject in need thereof    comprising the step of administering to a subject having a cancer    classified or diagnosed as having poor prognosis or being malignant,    using the method of any one of paragraphs 24-61, or the assay of any    one of paragraphs 62-79, at least one metabolic inhibitor in a    pharmaceutically acceptable carrier.-   98. The method of paragraph 97, wherein the at least one metabolic    inhibitor is an siRNA or antisense RNA agent.-   99. The method of any one of paragraphs 97-98, wherein the at least    one metabolic inhibitor specifically inhibits a metabolic malignancy    associated response signature biomarker set forth in SEQ ID NOs: 46,    49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and 153.-   100. A system for obtaining data from at least one sample from a    subject having a cancer, the system comprising:    -   a. a determination module configured to receive said at least        one sample from a subject having a cancer and perform an        expression analysis of ten or more malignancy associated        response signature biomarkers set forth in SEQ ID NOs: 1-154 on        said at least one sample to generate an expression data output;    -   b. a storage device configured to store said expression data        output from said determination module;    -   c. a comparison module configured to receive said expression        data output of the sample from the subject having a cancer and        perform at least one expression analysis on said expression data        output to determine the presence or absence of one of the        following conditions and produce a comparison data output:        -   i. the sample from the subject having a cancer has increased            expression of ten or more metabolic malignancy associated            response signature biomarkers set forth in SEQ ID NOs:            1-154; or        -   ii. the sample from the subject having a cancer does not            have increased expression of ten or more metabolic            malignancy associated response signature biomarkers set            forth in SEQ ID NOs: 1-154; and    -   d. an output or display module for displaying a content based in        part on the comparison data output from said comparison module,        wherein the content comprises a signal indicative that the        sample from the subject having a cancer has increased expression        of ten or more metabolic malignancy associated response        signature biomarkers set forth in SEQ ID NOs: 1-154, or a signal        indicative that the sample from the subject having a cancer does        not have increased expression of ten or more metabolic        malignancy associated response signature biomarkers set forth in        SEQ ID NOs: 1-154.-   101. The system of paragraph 100, wherein the content displayed on    said display module further comprises a signal indicative of the    subject being recommended to receive a particular treatment regimen.-   102. A method of identifying a candidate therapeutic agent against a    cancer initiating cell comprising the step of    -   a. exposing a BPLER cell culture to a test agent, wherein said        BPLER cell culture comprises human breast primary epithelial        cells (BPE) transformed with a defined set of genetic elements;    -   b. measuring expression of at least ten of the 154 malignancy        associated response signature biomarkers of paragraph 1 in the        culture,    -   c. comparing the expression of the same at least 10 biomarkers        of malignancy associated response signature biomarkers of        paragraph 1 as was measured in step (b) to an expression        signature reference from a BPLER cell culture that has not been        exposed to the test agent, wherein a decrease of expression of        at least 5 of the at least 10 genes in the test culture compared        to the expression signature reference indicates that the test        agent is a candidate therapeutic agent against a cancer        initiating cell.-   103. A therapeutic agent identified by the method of paragraph 102.-   104. The therapeutic agent of paragraph 103, wherein said agent is    an siRNA agent, an antisense RNA agent, an antibody or    antigen-binding fragment thereof, or a small molecule compound.-   105. A pharmaceutical compound comprising the therapeutic agent of    paragraph 103 and a pharmaceutically acceptable carrier.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology, andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 18th Edition, published by Merck Research Laboratories, 2006(ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006. Definitions of common terms inmolecular biology are found in Benjamin Lewin, Genes IX, published byJones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew etal. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982);Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989);Davis et al., Basic Methods in Molecular Biology, Elsevier SciencePublishing, Inc., New York, USA (1986); or Methods in Enzymology: Guideto Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. KimmerlEds., Academic Press Inc., San Diego, USA (1987); Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et. al., ed., John Wiley and Sons, Inc.) and Current Protocolsin Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons,Inc.), which are all incorporated by reference herein in theirentireties.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that could beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

EXAMPLES Example 1

BPLER and HMLER Breast Cancer Cells Retain Progenitor-Like andMyoepithelial-Like Features, Respectively

It has been shown that different populations of human breast primaryepithelial cells (BPE and HME), selected in chemically-defined media(e.g., WIT-T and MEGM, respectively), give rise to distinct tumorphenotypes upon transformation with the same set of genetic elements(e.g., hTERT, SV40 early region and h-RAS^(V12)), indicating that keyproperties of a tumor can be predetermined by its cell of origin. BPEtransformed derivatives (BPLER) are highly malignant, whereas HMEtransformed derivatives (HMLER) are poorly tumorigenic compared to BPLERcells, although the two cell lines proliferate at a similar rate invitro. As described herein, we have determined that such properties canbe associated with a different state of differentiation.

To exclude any effects related to cell culture medium or substrate, allexperiments described herein were performed by propagating BPLER andHMLER cells in the same medium (WIT-T) in standard tissue cultureplates, which did not alter the relative rate of proliferation. Underthese conditions, BPLER cells expressed a wide array of luminal andmyoepithelial cytokeratins, including CK5, CK14, CK17, CK8, and CK18,consistent with an epithelial progenitor-like phenotype, whereas HMLERcells expressed lower levels of most cytokeratins except CK5.Accordingly, BPLER cells retained an epithelial morphology andco-expressed CK14 and CK18 proteins at the single-cell level, a propertyof mammary epithelial progenitors and terminal duct lobular unit (TDLU)cells, from which breast cancer is believed, without wishing to be boundor limited by theory, to originate. BPLER cells also expressedE-cadherin and Vimentin transcripts, whereas HMLER cells expressedhigher levels of vimentin but lower levels of E-cadherin transcripts.Both cell lines were triple-negative (ER−, PR−, HER−) and expressed EGFRmRNA.

Together, these data demonstrate that BPLER cells retain aprogenitor-like phenotype, expressing both luminal and myoepithelialmarkers, whereas HMLER cells retain a more committed myoepithelialphenotype. These findings confirm at a protein level previousmicroarray-based studies, which showed a mixed luminal/myoepithelial andmyoepithelial-like transcriptional profile associated with BPLER andHMLER cells, respectively.

BPLER Cells have Properties of Basal-Like Breast Tumor-Initiating Cells.

The phenotypic features of BPLER cells are similar to poor prognosishuman basal-like breast tumors (BL-BTs), which are enriched in breasttumor-initiating cells (BT-ICs). We found that BPLER cells retained hightumor-initiating potential, demonstrated by their ability to form tumorsin mice with as few as 50 cells. In contrast, we found that HMLER cellsdid not initiate tumors in mice with 5×10⁴ or fewer cells, indicatingthat most HMLER cells had shed their tumor-initiating potential.

The bulk of BPLER cells displayed a CD44⁺/CD24^(low/−/)ESA⁺ antigenicconfiguration, which was previously associated with human BT-ICs.However, we show that these markers are not sufficient to specify thetumor-initiating state, because poorly malignant HMLER cells alsodisplayed a similar phenotype.

To define clinical parameters, histological features of tumorsoriginated from BPLER cells were first analyzed. BPLER xenograftsstained positive for CK5 and CK14 and were negative for ER, were highlyproliferative (as determined by Ki-67 staining), had a high mitoticindex (as determined by phospho-Histone H3 staining), displayed a clearinflammatory infiltrate, a discernible tumor stroma, pushing borders,and had alternated areas of glandular focal differentiation with poorlydifferentiated areas, all of which are characteristic properties ofhuman basal-like tumors. Further, only BPLER cells that establisheddirect contact with the stroma retained vimentin expression, which wasundetectable in the remaining cells, i.e., BPLER cells not having directcontact with the stroma. Because vimentin is typically repressed indifferentiated epithelial cells, this indicates that the tumor stromaplays a role in maintaining the relatively uncommitted state of BPLERcells in vivo. Accordingly, BPLER cells give rise to tumors closelyresembling human basal-like tumors at the histological level.

To further corroborate these findings, we compared the globaltranscriptional profile of six different BPLER tumor explants, derivedfrom either Nude or NOD/SCID mice, with a set of 337 human primarytumors of known subtype (UNC337 dataset). To reduce background signalderived from human stroma, as opposed to BPLER mouse stroma, we adopteda principal component (PC) approach. All BPLER tumor explants clusteredwith human primary triple-negative breast tumors, and not with luminal,normal, or HER-2 subtypes. We further validated these findings using anindependent set of 47 human primary breast tumors (Richardson-06dataset), in which BPLER tumor explants also clustered withtriple-negative tumors.

Accordingly, as demonstrated herein, BPLER cells not only haveproperties of BT-ICs, but also initiate tumors with global features ofhuman primary basal-like breast tumors, thus representing a model ofbasal-like BT-ICs.

A Genome-Wide siRNA Screen Identifies Factors Required for Survival ofBasal-Like BT-ICs

The data described herein so far demonstrate that BPLER and HMLER cellsretain high and low tumor-initiating potential, respectively. Moreover,BPLER cells display several features of human basal-like breast cancer,including malignancy, cytokeratin expression, hormone receptor statusand CD44/CD24 antigenic configuration, and form tumors closelyresembling human basal-like breast cancer.

To identify survival factors associated with the tumor-initiating state,we performed a genome-wide siRNA lethality screen using BPLER and HMLERcell lines, which were derived from the same patient, harbored the sameset of genetic elements, and proliferated at a similar rate, but weredifferentially malignant. By silencing one gene at a time, acomprehensive overview of the key mechanisms supporting the growth ofhighly malignant basal-like breast cancer cells was obtained, andrevealed novel diagnostic biomarkers and therapeutic targets for moreeffective targeted diagnosis of and therapies against basal-like breasttumors.

The screen was organized into separate modules, each of which wasdeveloped independently. The assay was optimized to identify candidategenes required for survival of either BPLER or HMLER cells in anunbiased way on a genome-wide scale. We used siRNA libraries including17,378 siRNA pools, each containing 4 distinct siRNAs targeting the samegene, which were screened in triplicate for each cell line.

A total of 1025 siRNA pools decreased BPLER viability, of which 780 alsodecreased HMLER viability. These siRNAs include hits not associated withmalignancy but more generally affecting proliferation or survival, theglobal silencing of which has a greater likelihood of being cytotoxic toall cell types. For example, the top 20 hits in this category includedPLK1, KIF11, RRM2, BCL-XL and POL2RA, all of which are well-knownregulators of basic cellular functions (Table 1).

The remaining 245 pools were highly, moderately, and modestly selectivesiRNAs preferentially inhibiting BPLER cells as opposed to HMLER cells,and were therefore associated with tumor-initiating potential (Tables2A-2C).

To further increase confidence in these hits, a secondary screen wasperformed (termed herein as a “cherry-pick screen”) transfecting the 4siRNAs comprising each pool individually. Hits were confirmed if atleast one out of four siRNAs scored positive in the secondary screen,since several siRNA pools used in the primary screen are predicted tocontain only one functional siRNA. A total of 154 hits were confirmed inthe cherry-pick screen, where validation rates were 88%, 75% and 52% forhighly, moderately, and poorly selective hits, respectively (Table 3).These 154 high-confidence hits represent genes on which BPLER cellsselectively depend for survival, and therefore comprise a functionalmodule associated with tumor-initiating potential, referred to herein asa “Triple Negative Breast Cancer Gene Signature” (TGS) or a“Malignancy-Associated Response Signature” (MARS), as termed herein.

Poor Prognosis Human Breast Primary Tumors are Enriched for GenesRequired for BPLER Survival

Having identified genes selectively required for survival of BPLERcells, we next determined that such genes were enriched in malignanthuman primary breast tumors. A single sample GSEA approach was used toproject the TGS (MARS) gene set across a set of 47 human breast primarytumors and normal breast tissues (DFHCC3 dataset). A significantpositive correlation was observed between the expression of TGS (MARS)genes and the primary tumor expression profiles (p<1.2e-05), indicatingthat most genes were enriched in malignant breast lesions compared tonormal breast tissue.

Next, we assessed whether TGS (MARS) expression was specificallyassociated with poor prognosis breast tumors, which are enriched inBT-ICs, in an independent set of 295 human breast primary tumors withannotated clinical and molecular subtype data (NKI dataset). TGS (MARS)expression positively correlated with shorter survival (p<0.003),demonstrating that highly malignant cancer cells within primary tumorsretain the expression of genes required for survival of BPLER cells.

To define whether basal-like primary tumors, in particular, wereenriched in TGS (MARS) genes, we assessed their expression in thedifferent subtypes of breast cancer within the NKI dataset. TGS (MARS)genes were retained in highly malignant basal-like breast cancers, ascompared to HER-2, luminal A, and “normal” breast cancers (p<2.61×10⁻⁵).Notably, TGS (MARS) genes were also enriched in Luminal B tumors(p<3.79×10⁻⁶). These tumors comprise ˜15% of human breast tumors andhave variable prognosis. Luminal B tumors are ER positive, much likegood prognosis luminal A tumors, but are also moderately differentiated,resembling poor prognosis basal-like tumors. We next determined whetherTGS (MARS) enrichment separates tumors with different prognosis withinthe luminal B category. Impressively, two luminal B patient subgroupswith markedly different survival were identified based on TGS (MARS)expression. No significant differences in survival were observed withinthe other subtypes using the same approach. These data furtherdemonstrate the association between TGS (MARS) and malignancy, whichappears, without wishing to be bound or limited by theory, to beindependent from the tumor subtype.

Metastasis is the leading cause of death in breast cancer patients. Wenext determined whether metastatic tumors were also enriched in TGS(MARS) genes. We found that within the NKI dataset, patients with higherTGS (MARS) expression developed metastasis significantly earlier thanpatients with TGS expression (p<0.01). To further confirm these results,we performed an analysis of TGS (MARS) expression in 560 primary breasttumors collated from three independent datasets (EMC286, MSK82, EMC192),for which metastatic relapse data were available. In this setting,patients with higher TGS (MARS) expression also had a significantshorter metastasis-free survival (p<0.002).

In conclusion, the expression of TGS (MARS) genes was retained in avariety of malignant disease conditions, confirming the clinicalrelevance of the genes identified in our screens described herein asnovel diagnostic and prognostic biomarkers and therapeutic targets.

BPLER Cells are Selectively Sensitive to Proteasome and GlycolysisInhibitor Drugs

To determine the biological functions of the TGS genes, their enrichmentin canonical pathways was assessed. The most significantly enrichedpathways included Proteasome Degradation, Glycolysis, TNFα/NFκBsignaling, IL-7 signaling, mRNA processing, Transcription Initiation, IDsignaling, and GPCR Class A Rhodopsin-like. An approach based, in part,on the Pathway Expansion Analysis (PEXA) of Tu et al. was used toestablish how these pathways are related to the TGS genes and identifythe underlying mechanisms that drive their observed effects. Thisanalysis leveraged pathway and interaction curation to identify ahigh-confidence core interaction network. The network from theseanalyses included a large number of TGS genes that interacted directlyat the protein level, or were functionally related, supporting acoherent biological relationship within TGS genes.

To establish which of these pathways correlated with the basal-likephenotype in breast cancer patients, and select biological pathways fortherapeutic intervention, we analyzed the expression of TGS-associatedpathways in human primary breast tumors. Indeed, Proteasome, Proteasomedegradation, Glycolysis, mRNA Processing, and TNF-alpha pathways wereselectively enriched in poor prognosis basal-like tumors compared togood prognosis luminal A tumors, but not IL-7 signaling, ID signaling,GPCR Class A Rhodopsin-like and Transcription Initiation. Notably,pathways enriched in basal-like primary tumors were also enriched inLuminal B tumors.

Indeed, the integration of genome-wide functional and expression dataallows us to identify and select biological pathways the inhibition ofwhich can be therapeutically effective in the context of basal-likebreast tumors, because they are required for survival of highlymalignant basal-like breast cancer cells, and are also enriched in humanprimary BL-BTs.

To further support these results, we further validated some of thesepathways functionally. Proteasome Degradation and Glycolysis pathwayswere particularly attractive because chemical inhibitors are available.Moreover, Proteasome Degradation was the pathway most highly representedin the TGS genes, and also the most connected, as the hits were allsubunits of the 20S or 26S proteasome complexes. These included PSMA1,PSMA2, PSMA3, PSMB4, PSMC1, PSMC3, PSMD2, PSMD7, and PSMD14. Moreover,other TGS genes, such as UBL5, NEDD1, NEDD8, ANAPC2, and ANAPC4, werealso involved in the ubiquitin-proteasome system. On the other hand,only two hits were associated with Glycolysis, PFKL and GAPDH pathwaysbut their enrichment in the TGS was highly significant. HK1, whichinitiates glucose catabolism through the same pathway, also scoredpositive in the primary screen, but could not be validated in thesecondary screen. Most of the screening hits in these pathways wereclassified as highly or intermediate BPLER selective inhibitors, andcould each be validated with 2-4 individual siRNAs in the secondaryscreen.

We used chemical inhibitor drugs to further confirm the selectiveresponse of BPLER and HMLER cells to inhibition of proteasomal activityor glycolysis in an RNAi-independent manner. Indeed, BPLER cells weremore sensitive to increasing doses of bortezomib (a proteasome inhibitordrug) and 3-bromo-pyruvic acid (BRPA, a chemical inhibitor ofglycolysis) relative to HMLER cells. Treatment with bortezomib decreasedproteasomal activity similarly in both cell lines, demonstrating thatBPLER cells were intrinsically more sensitive to transient proteasomalinhibition. Exposure to BRPA for 4 hours, when most cells were viable,decreased ATP levels in BPLER and HMLER cells by 90% and 60%respectfully, indicating, without wishing to be bound or limited bytheory, a higher dependence of BPLER cells on glycolysis for ATPgeneration. However, this might also indicate, without wishing to bebound or limited by theory, a differential ATP consumption rate betweenthe two cell lines. Notably, the exquisite sensitivity of BPLER cells tobortezomib and BRPA was drug-specific, because BPLER and HMLER cellsresponded similarly to treatment with the anthracycline doxorubicin.

To further validate the correlation between tumor-initiating potentialand response to either proteasome or glycolysis inhibition, we assessedthe dependency of untransformed BPE cells on these pathways. AlthoughBPE were as proliferative as HMLER and BPLER cells, they were resistantto both proteasome and glycolysis inhibitors, further confirming thespecificity of these drugs for highly malignant breast cancer cells.

While glycolysis inhibitors are in pre-clinical development, proteasomeinhibitors are already in the clinic for the treatment of multiplemyeloma and mantle cell lymphoma, and can be rapidly investigated forbreast cancer therapy in a clinical setting. Nonetheless, some phaseI/II clinical studies have shown poor response of unselected breastcancer patients to proteasome inhibitors. Accordingly, we furtheredexplored and determined the molecular basis of BPLER dependence on theproteasome degradation pathway for survival.

Proteasome Inhibition Selectively Promotes Noxa-Dependent Apoptosis inBasal-Like Breast Cancer Cells

The data described herein show that highly malignant BPLER cellsselectively depend on proteasomal activity for survival compared topoorly malignant HMLER cells and untransformed breast primary epithelialBPE cells. To further define factors underlying such properties of BPLERcells, we first determined the molecular mechanism by which proteasomeinhibition affects the growth of these cells. Treatment with clinicallyrelevant doses of bortezomib for 24 hours induced marked cleavage ofcaspase 3 and PARP in BPLER cells, but only partially in HMLER cells andnot in BPE cells, indicating selective activation of apoptosis in BPLERcells. Likewise, upon treatment with low-dose bortezomib, 34%, 17% and2% of BPLER, HMLER and BPE cells stained positive for Annexin V/PI,whereas co-treatment with the pan-caspase inhibitor ZVAD dramaticallyreduced the number of double-positive cells, further demonstrating theselective induction of caspase-dependent cell death in BPLER cells uponproteasome inhibition.

Other basal-like breast cancer cell lines, such as HCC-1143 andHCC-1937, were also exquisitely sensitive to proteasome inhibitioncompared to mesenchymal MDA-MB-231 and MDA-MB-436 or luminal MCF7 andBT-474 breast cancer cell lines. In fact, treatment with bortezomibdecreased cell viability by 50-65% in basal-like cells, but only by20-25% in mesenchymal cells and 5% in luminal cells. Moreover,bortezomib induced PARP cleavage in both HCC1143 and HCC1937 cells, butnot in MCF7 or MDA-MB-436 cells. Therefore, as shown herein, theselective response to proteasome inhibitors is an intrinsic feature ofhuman basal-like breast cancer cells, and not limited to BPLER cells.

BH3-only proteins have been shown to mediate bortezomib-inducedapoptosis in multiple myeloma cells, which are also exquisitelysensitive to inhibition of the proteasome. We assessed the levels ofthese proteins in BPLER cells upon treatment with bortezomib. Notably,the BH3-only protein Noxa was highly increased in treated cells, whereasother BH3-only proteins were only modestly increased (e.g., Bik, Bim),unchanged (e.g., Bid, Bad), or decreased (e.g., Puma).

Indeed, we determined that Noxa was necessary to mediate apoptosis inBPLER cells, because its silencing by RNAi partially rescued the lethaleffect of proteasome inhibition in BPLER cells, similar to thepan-caspase inhibitor ZVAD. On the other hand, silencing Bim or Bik didnot rescue BPLER cells from cell death, whereas silencing both Bik andNoxa simultaneously did not further increase cell viability uponproteasome inhibition. Finally, Noxa was also induced in thebortezomib-responsive cell lines HCC1143 and HCC1937 cells, and not inbortezomib-resistant luminal MCF7 cells, further confirming the centralrole of Noxa in mediating bortezomib-induced cell death. However, Noxadid not appear sufficient to activate apoptosis, because it wassimilarly induced in HMLER cells and MDA-MB-231 cells upon treatmentwith bortezomib. Although Noxa was barely detectable at the proteinlevel in all untreated cells, it was actively transcribed intriple-negative breast cancer cells compared to luminal breast cancercells and untransformed breast epithelial cells, indicating, withoutwishing to be bound or limited by theory, that triple-negative cellshave high ability to induce Noxa because of high baseline mRNA levels.Noxa mRNA was only modestly increased in BPLER and HMLER cells uponproteasome inhibition, despite a strong induction of Noxa protein inboth cell lines. Together, these findings demonstrate that basal-likebreast cancer cells undergo Noxa-dependent apoptosis upon proteasomeinhibition.

Proteasome Inhibitors Interfere with Mitosis in Highly MalignantBasal-Like Breast Cancer Cells.

The proteasome is a key regulator of cell cycle. We next determinedwhether the selective response of BPLER cells to proteasome inhibitorsis linked to an abnormal regulation of cell cycle dynamics. To definethe physiological response of normal breast epithelial cells toproteasome inhibition, we analyzed the cell cycle distribution of BPEcells treated with bortezomib for 24 hours, which showed a markedaccumulation of cells in G2/M phase. A similar response was observed inHMLER cells, and BPLER cells treated with suboptimal doses ofbortezomib. Accordingly, proteasome inhibition induced a marked increaseof p21 and p27 proteins, two main cell cycle inhibitors, in all thethree cell lines.

To exclude secondary effects of apoptosis, and track progression throughdifferent cell cycle phases, we analyzed cell cycle distribution uponproteasome inhibition in BPE, HMLER and BPLER cells in the presence ofthe pan-caspase inhibitor ZVAD, which inhibits apoptosis induced byproteasome inhibition, and nocodazole, which arrests cells as they reachmitosis.

All the three cell lines underwent mitotic arrest when treated withnocodazole for 24 hours. Addition of bortezomib delayed progression toG2/M phase in BPE cells and, to a lesser extent, HMLER cells, but not inBPLER cells. Notably, BPLER cells failed to arrest in G1 or G2 phasesand entered mitosis, upon proteasome inhibition, whereas BPE cells and,in part, HMLER cells remained in interphase, as determined bymorphology, Histone H3 phosphorylation and DNA staining. Likewise,HCC1143 basal-like breast cancer cells entered mitosis upon proteasomeinhibition.

Basal-like breast cancer cells are known to be highly sensitive toantimitotic agents. Because proteasomal activity drives exit frommitosis, we next determined whether proteasome inhibition interferedwith mitosis in BPLER cells. When apoptosis was blocked by ZVAD,proteasome inhibition delayed the exit from mitosis of BPLER cells thatwere first synchronized with nocodazole. Notably, a mitotic arrestachieved by nocodazole treatment for 24 hours was sufficient to activateapoptosis in BPLER cells, although not as much as proteasome inhibition,indicating that disruption of normal mitosis contributed tobortezomib-induced apoptosis. Indeed, bortezomib appeared to act as anantimitotic agent, but was more potent compared to nocodazole,presumably because of concomitant induction of proapoptotic factors suchas Noxa. Importantly, BPLER cells became resistant to bortezomibtreatment once they reached confluence, further supporting a highersensitivity of dividing cells to proteasome inhibitors.

Accordingly, these data show basic mechanisms of action of bortezomib inbasal-like breast cancer cells, which involve, in part, inactivation ofthe G2/M checkpoint and disruption of normal mitosis.

Proteasome Inhibition Inhibits the Outgrowth of Basal-Like Breast Tumors

Although breast cancer patients have previously been shown to respondpoorly to proteasome inhibitors, we next determined whether bortezomibexerts an antitumoral effect on basal-like breast cancer cells in vivo.For all the experiments described herein, bortezomib was given everythree days (q3d), reflecting clinical protocols used in human patients,at doses of 0.5 mg/kg or 1.0 mg/kg intraperitoneally (i.p.), which werepreviously reported to be well-tolerated in mice. We did not observe anysignificant weight loss after 10-16 days of therapy, at which timepoints mice were sacrificed.

We first assessed the effect of bortezomib on pre-establishedsubcutaneous BPLER xenografts in nude mice. The growth of BPLER tumorswas influenced by host factors, and tumors developed between 2 and 5weeks, even when high number of cells (5×10⁵) cell were injected. Toreduce experimental variability, we selected mice bearing tumors ofhomogenous size (˜0.5 mm in diameter after 3 weeks from injection),which were randomized into two groups. Each group received 6 doses ofeither vehicle (DMSO) or bortezomib at 0.5 mg/kg i.p. q3d over a 16-dayperiod, respectively. At the end of the treatment period, mice weresacrificed and tumor weight was assessed. Treatment with bortezomibsignificantly reduced median tumor weight by 54% after 16 days(p=0.001). Histological examination of residual tumors revealedextensive cleaved caspase-3 positive apoptotic/necrotic areas in bothtreated and untreated tumors, which is a typical feature of humantriple-negative breast tumors. We could not quantify with precision therelative effect of the treatment on apoptosis by immunohistochemistryalone. Although the histological picture was compatible with theinduction of cell death, without wishing to be bound or limited by atheory, other mechanisms of tumor growth inhibition can also play arole.

To minimize host-related factors in inducing response to bortezomib, wedecided to further validate these data in a syngeneic mouse model ofbasal-like breast cancer, where the influence of the immune system orother host factors is diminished.

In the course of our studies described herein, we identified asubpopulation of basal-like breast cancer cells within the 4T1 cellline, which derives from a spontaneous BALB/c mouse breast tumor. Thesecells, termed 4T1-E, display a CD44^(med)/CDH1^(low)/ESA^(high)epithelial phenotype, whereas the remaining cells, termed 4T1-M, displaya CD44^(high)/CDH1⁻/ESA^(low) mesenchymal phenotype. The two populationsare stable, have a similar proliferation rate, and do not appearhierarchically linked, because they did not convert into each otherafter extensive culture in vitro.

Indeed, 4T1-E cells expressed higher levels of epithelial markers,including CDH1, ZO-1, CK-14 and EGFR, and lower levels ofmyoepithelial/mesenchymal markers, including Vimentin, MMP3 and Snail,compared to both 4T1-M cells as well as 67NR cells, which originate froman established mesenchymal clone previously isolated from the sametumor. Moreover, 4T1-E over-express the murine stem-like markers CD49f,CD24 and CD29 compared to 67NR cells. Finally, 4T1-E cells are moretumorigenic than 4T1-M cells and 67NR cells when transplanted in themammary fat-pad of syngeneic mice, can form tumors with as few as 500cells in syngeneic BALB/c mice after 3 weeks with complete penetrance,and give rise to highly malignant CK-14 positive epithelial breasttumors. Indeed, 4T1-E had a stem/progenitor-like phenotype, andrecapitulated several features of BPLER cells and human basal-likebreast tumors. 4T1-E also responded to bortezomib in vitro. Therefore,we assessed the therapeutic effect of bortezomib on the outgrowth of4T1-E tumors in syngeneic BALB/c mice. Because all tumors grew veryrapidly in this model, treatments were started 2 days after tumor cellinoculation. Similarly to BPLER xenografts, treatment with bortezomib (4doses at 1.0 mg/kg i.p. q3d) reduced the median tumor weight by 56%after 10 days of treatment (p<0.01).

In conclusion, bortezomib exerts a significant therapeutic effect onbasal-like tumors in vivo, although it does not induce completeremission of the tumors. Thus, in some embodiments of the methodsdescribed herein, a patient determined to have increased or enrichedexpression of at least one, or more preferably, at least 10 TGSbiomarker genes in a tumor sample using the methods described herein, ora patient diagnosed having a basal-like breast tumor, can be furtheradministered a proteosome inhibitor. In some such embodiments, theproteosome inhibitor is bortezomib.

Recent studies on animal models have indicated that basal-like breasttumors derive from transformed epithelial progenitors expressing bothluminal and myoepithelial markers. Herein we demonstrated that humanprimary epithelial progenitor-like cells (BPE) give rise to tumorsclosely resembling human BL-BTs upon transformation with canonicaloncogenes. Our data described herein indicate that epithelialprogenitors retain the potential to initiate such tumors upontransformation. This is not an artifact of in vitro transformation,because introduction of the same genetic elements intomyoepithelial-like cells (HME) did not induce the development ofbasal-like tumors. Moreover, the oncogenes used to transform BPE cellsrecapitulate common genetic alterations occurring in human BL-BTs,including loss of function of p53 and Rb (which are inactivated by SV40)and activation of RAS signaling. As shown herein, transformation ofhuman primary cells into basal-like tumor-initiating cells appearsdependent on the cell of origin. However, it is possible that othercombinations of oncogenes can also induce basal-like phenotypes fromdifferent cells of origin.

BPLER cells represent a novel and the first genetically-defined model ofa tumor-initiating cell with a basal-like phenotype. According to thecancer stem cell (CSC) hypothesis, only a minor subpopulation of cancercells within a tumor retain tumor-initiating potential, whereas the bulkof tumor cells undergoes differentiation, thus shedding malignantproperties. The CSC model can apply to certain low-grade tumors(including, e.g., luminal A breast tumors), in which cancer cells arecapable of differentiation, however, human basal-like breast tumors aretypically poorly differentiated lesions with a diffuse progenitor-likephenotype. In other tumors, where differentiation is impaired by geneticalterations, such as loss of p53, such as, for example, human basal-likebreast tumors, the majority of cancer cells retain malignant potential.Our data demonstrate that human progenitor-like epithelial cellsexpressing BT-IC markers give rise to basal-like epithelial tumors,showing that at least certain stem-like features, such as self-renewal,can be acquired by epithelial progenitors as a result of transformation.

Previous studies have shown that passage through anepithelial-mesenchymal transition (EMT) generates mesenchymal cells withcancer stem-like properties. The pure mesenchymal phenotype does notappear to be a corollary of breast cancer “stemness,” because BPLERcells retain both epithelial and mesenchymal features (i.e., ametastable phenotype) and are highly malignant, similar to 4T1-E mousecells. Mesenchymal breast cancer cells have been shown to initiatetumors resembling claudin-low tumors or other rare mesenchymal breastcancer variants, but not basal-like epithelial tumors.

It should be noted that, since most of the established EMT markers areeither luminal or myoepithelial transcripts, the metastable (mixed)phenotype is indicative of a progenitor-like state of differentiation.Therefore, metastable cells can represent early epithelial progenitors.

Unbiased forward genetic screens have been powerful tools with which toidentify molecules involved in fundamental pathways in model organisms,but have been difficult to do in mammalian cells. The discovery of RNAi,coupled with the sequencing of the genome, has provided the opportunityto identify candidate genes required for a biological process in anunbiased way on a genome-wide scale by silencing one gene at a time. Asdescribed herein, we performed a genome-wide siRNA lethality screen todefine functional vulnerabilities associated with the tumor-initiatingstate in the BPLER/HMLER system. We identified 154 therapeutic targetsthat can be harnessed for diagnosis, prognosis, and treatment ofcancers, such as basal-like breast tumors. These targets are clinicallyrelevant because they are enriched in human primary basal-like breasttumors and their expression correlates with poor prognosis andmetastatic relapse. The prognostic value of these biomarker genes doesnot depend on their direct association with basal-like tumors, becausethey also discriminate between good and poor prognosis patients withinthe luminal B category.

In contrast to other breast cancer signatures, we have identified genesand pathways that are not only predictive of tumor subtype, prognosis,and risk of metastasis, but are also required for survival of basal-likeBT-ICs in vitro. Importantly, the malignancy associated responsesignature genes described herein can be used to identify breast cancerpatients who are at higher risk of death or metastasis, and who are morelikely to respond to biological therapies targeting MARS-related genesor networks of genes.

Amongst the biological pathways selectively inhibiting BPLER cells, wefocused, in part, on the proteasome-ubiquitin system, not just becauseit was one of the most significantly enriched for pathways, but alsobecause proteasome inhibitor drugs are already in the clinic formultiple myeloma and relapsed mantle cell lymphoma. Although bortezomibis the only proteasome inhibitor approved for clinical use, 5second-generation proteasome inhibitors are currently in phase I trials.Moreover, 10 drugs targeting specific E1-activating enzymes andE3-ubiquitin ligases are also in clinical development. It is believedthat proteasome/ubiquitination inhibitors represent a novel class ofdrugs, whose number of potential targets may exceed protein kinases. Ofnote, BPLER cells also selectively responded to glycolysis inhibitors,which are currently in preclinical development. Accordingly, in someembodiments of the methods described herein, a subject having increasedor enriched expression of at least one or more, preferably, at least 10,MARS (TGS) biomarker genes of SEQ ID NOs: 1-154 can be treated using anyproteosome inhibitor, such as bortezomib and second-generationproteasome inhibitors in development, as well as drugs targetingspecific E1-activating enzymes, E3-ubiquitin ligases, and inhibitors ofglycolysis.

Treatment with bortezomib induced apoptosis in basal-like breast cancercells, but not in luminal or mesenchymal breast cancer cells or innormal breast epithelial cells. Based on our data described herein, atleast two mechanisms underlie such selectivity. First, basal-like cellsare poised to undergo bortezomib-induced apoptosis, compared to luminalcells, because of active transcription of the proapoptotic factor NoxamRNA, whose protein levels are kept in check by the proteasome. Second,consistent with their malignant nature, basal-like breast cancer cellsare unable to activate cell cycle checkpoints upon proteasomeinhibition, which represents the physiological response of normal breastepithelial cells. Indeed, proteasome inhibition interferes with mitosisexit, which represents a pro-apoptotic signal per se, because BPLERcells also responded to antimitotic agents.

Alteration of the G2/M checkpoint is a hallmark of human BL-BTs, forwhich treatment with antimitotic agents represents the standard of care.Bortezomib acted as an antimitotic agent by interfering with mitosisexit, but also induced the expression of potent pro-apoptotic moleculeslike Noxa. Significantly, proteasome inhibition was more effective thaninduction of mitotic arrest alone in eliminating basal-like breastcancer cells.

Little is known about the mechanisms linking mitotic arrest toapoptosis. It has been proposed that prolonged mitotic stall inducesinactivating phosphorylation of antiapoptotic BCL2-family proteins,including BCL2, BCL-XL and MCL1. Interestingly, we observedphosphorylation of MCL1 (a main antagonist of Noxa) in mitotic BPLERcells treated with bortezomib, which can contribute to induction ofapoptosis upon proteasome inhibition.

Recent studies have shown that targeting mitosis exit by inhibitingCDC20, which links proteasomal activity with mitosis progression, leadsto tumor regression in vivo. Our data demonstrate that proteasome-basedtherapies are effective against basal-like tumors in vivo as well. Thesedate are in agreement with eight phase I/II studies assessingchemotherapy combination regimens with bortezomib, which showed partialresponse rates of 10-20% in unselected breast cancer patients.

Provided herein are novel insights into mechanisms of resistance inhuman cancer cells. We showed herein that non-dividing BPLER cellsbecame resistant to bortezomib. However, six out of eight clinicalstudies assessing the effect of bortezomib on breast tumors were basedon combination regimens with other drugs, such as doxorubicin, that areknown to induce G1 arrest. Interestingly, administration of bortezomibin combination with docetaxel, which induces mitotic arrest, in onestudy compared favorably with patients treated with docetaxel alone.

On the other hand, our data indicate that basal-like tumors, but notluminal tumors, are more likely to respond to proteasome inhibitors.Because luminal tumors comprise over 70% of human breast cancers, atargeted approach aimed at identifying and selecting patients, based onexpression of at least one, but more preferably at least 10 MARSbiomarker genes, with basal-like tumors can increase response rates toproteasome inhibitors in the clinic. In conclusion, we identified coresurvival pathways on which basal-like BT-ICs depend for survival, andidentified novel diagnostic, prognostic, and therapeutic biomarkertargets with documented clinical relevance. Proteasome inhibitionprovides one therapeutic strategy to selectively tackle basal-likebreast tumors. Other therapeutic approaches, such as glycolysisinhibition, can also be effective against poor prognosis breast tumors.

Example 2

Triple negative breast cancers (TNBC) are poor prognosis cancers,frequently associated with TP53 mutation and active RAS signaling. Asdescribed herein, we show that human progenitor-like primary mammaryepithelial cells (BPE) selectively acquire TNBC-initiating propertiesafter transformation with defined genetic elements. A genome-wide siRNAscreen identified 154 genes upon which BPE transformed derivatives(BPLER) selectively depend for survival, compared to geneticallyidentical myoepithelial-like cells from the same donor (HMLER). BPLERselective dependencies comprise a malignancy associated responsesignature (MARS) or TNBC gene signature (TGS) whose expressioncorrelates with poor prognosis and metastasis in breast cancer patients.The MARS is enriched in proteasome-ubiquitin system, metabolism, RNAsplicing, mitosis and molecular transport related genes. BPLER and humanepithelial TNBC cell lines are poised to accumulation of the Mcl-1antagonist Noxa and selectively depend on Mcl-1 for survival. In fact,proteasome inhibitor drugs target Mcl-1 dependency in these cells. Using“chemogenetics” to compare the MARS with gene expression signaturesinduced by >1,300 chemicals, we also identified histone deacetylaseinhibitor drugs as selectively cytotoxic for BPLER. Thus, as describedherein an unbiased siRNA screen identified novel classes of candidatedrugs to treat TNBC.

Triple-negative breast cancers (TNBC), defined by their lack of estrogen(ER) and progesterone (PR) receptor and Her2 expression, are anespecially aggressive group of tumors often found in young Afro-Americanand Hispanic women, with the shortest survival of breast cancersubtypes. TNBC, which comprise ˜15% of human breast tumors, areassociated with BRCA1 mutations and/or Tp53 and Rb loss of function inover 90% of cases¹. Compared to ER+ luminal tumors, TNBCs are poorlydifferentiated and most cells have features of epithelial progenitorcells.^(2,3) Targeted genetic ablation of Brca1, Tp53 and/or Rb inmammary epithelial progenitor cells in mice leads to TNBC-likemalignancies, suggesting that TNBC arise from transformed epithelialprogenitor cells⁴⁻⁶. TNBC generally express markers associated withbreast tumor-initiating cells⁷⁻⁹, CD44⁺/CD24^(low/−)/ESA⁺, EGFR andvaried combinations of cytokeratins CK5, CK14, CK17, CK8, CK18². Thesetumors rapidly become resistant to chemotherapy and are refractory tohormonal therapy and HER-2 inhibitors¹.

As described herein, we performed an unbiased genome-wide siRNAlethality screen to identify factors on which epithelial TNBC-initiatingcells selectively depend for survival. For the screen we took advantageof a method to generate pairs of essentially genetically identicalbreast cancer cell lines, BPLER and HMLER, by transforming normal breastprimary epithelial cells with defined genetic elements¹⁰ (FIG. 1A). Wefound that BPLER cells, which derive from progenitor-like epithelialcells, have the phenotype of TNBC and retain TNBC-initiating potential,uniformly generating tumors that resemble primary TNBCs after injectionof just 50 cells in immunodeficient mice, while HMLER cells aremyoepithelial-like and do not form tumors after injection of 5×10⁴cells. Thus BPLER cells are a good model for studying malignant traitsof TNBC. Knockdown of 154 genes selectively reduced the survival ofBPLER cells compared to genetically related HMLER cells prepared fromthe same donor. These hits were highly enriched for components of theproteosome and for genes that participate in the ubiquitin-proteosomesystem (UPS) as well as for genes important in RNA splicing, glycolysis,DNA replication and mitosis. The 154 hits constitute a malignancyassociated response signature (MARS) or TNBC gene signature (TGS) ofgenes whose expression is associated with reduced survival and earlymetastasis in human primary breast tumors. The MARS predicted that TNBCwould be selectively vulnerable to proteosome and histone deacetylationinhibition. Indeed BPLER and TNBC cell lines were highly susceptible tothese classes of drugs, providing novel approaches to treat this deadlydisease that is largely refractory to current therapeutic regimens.

Results

BPLER Cells are Tumor-Initiating Epithelial Cells that Give Rise to TNBC

At least two types of breast epithelial cells, termed BPEC and HMECcells, can be reproducibly derived from normal human breast organoidsexpanded in vitro using different chemically-defined media, WIT andMEGM, respectively¹⁰ (FIG. 1A). BPEC have a progenitor-like phenotype,whereas HMEC have a myoepithelial-like phenotype. After transformationwith hTERT, SV40 early region and H-RAS^(V12) in these respective media,they give rise to BPLER and HMLER cancer cell lines. Because these cellsare essentially genetically identical but differentially tumorigenic,they represent an attractive model to study epigenetic changesassociated with tumor initiation. To exclude continuing contributions ofthe differences in the growth media to their tumor properties, wepropagated both cell lines in WIT medium for two weeks before analyzingtheir phenotype, proliferation and tumor-forming capacity. (Thephenotypic properties of the HMLER cell line once established did notchange upon culture in WIT medium; therefore all subsequent experimentscompared BPLER and HMLER cells both grown in WIT medium.) We first setout to determine whether either of these cell lines, which are bothtriple negative and EGFR+ (FIG. 1B), might be a good model for TNBC.Both BPLER and HMLER also stain with markers CD44+CD24^(low/−)ESA+ usedto define breast tumor-initiating cells (BT-IC) (FIGS. 8A-8C). AlthoughBPLER and HMLER cells proliferated at a similar rate in vitro (FIG. 1C),BPLER cells uniformly formed tumors in mice with as few as 50 cells,whereas 5×10⁴ or fewer HMLER cells did not (FIG. 1D). Thus only BPLERare highly enriched for breast tumor-initiating cells (BT-IC). When morecells are injected, HMLER form squamous tumors that do not resemble anyof the common breast cancer subtypes¹⁰.

BPLER expressed a wide array of luminal and myoepithelial cytokeratinmRNAs, as is typical of TNBCs and untransformed bipotent epithelialprogenitor cells, and intermediate levels of both E-cadherin andvimentin mRNAs, whereas HMLER cells had reduced expression of luminalcytokeratin and E-cadherin mRNA and expressed higher levels of vimentinmRNA (FIG. 1E, FIG. 8C). When examined by immunofluorescence microscopy,BPLER, but not HMLER, cells co-expressed CK14 and CK18 proteins, aproperty of mammary luminal epithelial progenitors². Therefore, althoughboth BPLER and HMLER cells were triple-negative and expressed cellsurface markers that have been used to identify BT-IC, only BPLER cellshad tumor-initiating potential and expressed markers of TNBCs andepithelial progenitors.

Next we asked whether tumors formed in vivo by subcutaneous injection ofBPLER cells in immunodeficient mice resemble human primary TNBCs. BPLERtumor explants stained positive for CK5 and CK14 and negative for ER,had a high mitotic index and displayed a clear inflammatory infiltrate,reactive tumor stroma and pushing borders, all of which are typical ofhuman TNBC^(1,11). Moreover, BPLER tumors were poorly differentiatedepithelial lesions, with focal areas of glandular differentiation,consistent with their epithelial phenotype¹⁰. To examine more closelywhether BPLER tumors resemble TNBCs, we compared by unsupervisedhierarchical clustering the global transcriptional profile of six BPLERtumor explants that grew in either nude or NOD/SCID mice with mRNAexpression profiles of 337 human primary breast tumors of known subtype(UNC337 dataset¹²). The BPLER tumor explants clustered with humanprimary TNBC and not with luminal A/B, normal-like or HER2+ subtypes.These findings were verified by analyzing an independent set of 7 normalbreast samples and 40 human primary breast tumors (Richardson-06dataset¹³), classified as basal-like or non-basal-like. BPLER againclustered with basal-like tumors, the most common gene expressionsubtype of TNBC. Thus, BPLER initiate tumors closely resembling humanTNBCs and are a good model for studying TNBC.

A Genome-Wide siRNA Screen Identifies BPLER Survival Dependency Factors

Although BPLER and HMLER are essentially genetically identical, theyimportantly differ in their differentiation state and ability to formtumors. To identify functional vulnerabilities associated with aTNBC-initiating phenotype, we performed a high throughput genome-widesiRNA lethality screen by transfecting BPLER and HMLER cell linesderived from the same donor in triplicate wells, each with a pool of 4siRNAs targeting distinct sites within a single gene using the DharmaconsiGenome library of 17,378 gene targets (FIG. 2A). Three days later themean number of surviving cells was assessed by CellTiterGlo assay andthe ratio (R) of viable BPLER to viable HMLER cells was calculated. Datadescribing the optimization of the screen are described in FIGS. 9A-9G.The majority of the pools scored within one median absolute deviation(MAD) of the plate median. 1025 siRNA pools significantly decreasedBPLER viability, of which 780 decreased HMLER viability to a similarextent (Table 1). Of the remaining 245 pools, 26 were highly selectivelylethal for BPLER(R≦0.55), 76 were moderately selective (0.55<R≦0.65) and143 were modestly selective (0.65<R≦0.75) (FIG. 2B, Tables 2A-2C). Tovalidate the candidate hits (i.e. genes selectively required for BPLERsurvival), we next screened each of the hits by transfecting BPLER andHMLER cells separately with each of the four individual siRNAscomprising each pool. In the validation screen, 154 of the pools (63%)reconfirmed with at least one siRNA (Tables 2A-2C, FIG. 2B). Notsurprisingly, the validation rates were higher for hits with lower Rvalues (88% of highly selective, 75% of moderately selective and 52% ofmodestly selective hits were validated) (FIG. 2C).

The BPLER Dependency Genes are Highly Expressed in TNBC and theirExpression is Associated with Poor Prognosis in Breast Cancer

To evaluate the clinical relevance of the 154 validated BPLER dependencygenes, we examined the expression of this gene signature across breastcancer subtypes using the NKI mRNA expression profiles of 295 primarybreast cancers, classified as basal-like, HER2+, luminal A, luminal B,or normal-like¹⁴. The dependency genes were significantly over-expressedin both basal-like and luminal B tumors relative to other subtypes(p-value <3×10⁻⁵ and 4×10⁻⁶, respectively, by Kolmogorov-Smirnov (KS)test). When the analysis was limited to the 23 highly selective genehits, the same two subtypes significantly overexpressed these genes(p<3×10⁻⁶ and 4×10⁻⁵) (FIG. 2D). To determine whether overexpression ofthese genes might correlate with clinical prognosis, we divided the NKItumors into two groups based on their expression of either the 154 orthe subset of 23 highly selective BPLER dependency genes. In both cases,patients with high expression of the dependency genes had significantlyreduced survival. The survival difference was significantly greater ifthe analysis were restricted to the highly selective hits (FIG. 2E).Because high expression of the 154 BPLER dependency genes is linked toTNBC and to poor prognosis, we consider the 154 genes a malignancyassociated response signature (MARS) or TNBC gene signature (TGS).Luminal B tumors, which comprise ˜15% of human breast tumors, havevariable prognosis. Within this category, expression of the TGS orhighly selective hits clearly separated luminal B tumor patients withpoor survival (p<0.003 and p<0.01, respectively), indicating that theMARS is enriched in highly malignant breast tumors, independently oftumor subtype (FIG. 2F). Indeed, it can be used to predict prognosis inluminal B tumor patients. Within the NKI dataset, patients with higherMARS or highly selective hit expression also were diagnosed withmetastasis significantly earlier than patients with low MARS expression(FIG. 2G). A highly significant association of low MARS expression withmetastasis-free survival was also found by analyzing an independentdataset of 286 primary breast tumors (EMC286 dataset^(15,16), FIG. 10).In conclusion, BPLER dependency genes are more highly expressed inhighly malignant human primary breast cancers, and might serve astherapeutic targets against TNBC.

BPLER Dependency Genes are Enriched for Proteasome Subunits and GenesInvolved in Mitosis, Metabolism, DNA Transcription and RNA Biogenesis

An examination of the 23 validated highly selective BPLER dependencygenes (FIG. 2C) indicated to us that BPLER cells might be selectivelydependent on proteasome function since 7 of these genes are proteasomecomponents (hypergeometric p-value 1.1×10⁻¹⁴) and an additional hit(UBL5) encodes for a ubiquitin-like molecule. Another group of 4 genesis involved in mRNA biogenesis and nuclear export. These included thespliceosome component PRPF8 and 2 RNA helicases (DDX19B, DHX8) and thenuclear RAS protein RAN, all involved in mRNA nuclear export. Inaddition the hit list contained 4 Zinc finger genes (ZNF490, ZNF574,ZNF643, FIZ1), all likely transcriptional activators, and a nuclearreceptor coactivator gene SNW1 associated with activation of retinoicacid and estrogen-regulated genes. Also of note were 2 genes RACGAP1 andHAUS3 that regulate the mitotic spindle and PP2CA, the gene encoding thecatalytic subunit of the tumor suppressor PP2A that modulates the actionof many oncogenic protein kinases.

To begin to make sense of the larger 154 member MARS, we first looked atwhether the hits could be grouped into well-defined functionalcategories, using a combination of the pathways and processes in theReactome¹⁷, KEGG¹⁸ and Wikipathway¹⁹ databases. Of the 154 MARS genes,121 genes have well described annotations that could be grouped into 13functions with at least 3 genes assigned to each function (Table 4A).The proteasome was highly over-represented in the MARShit list with 10proteasome subunits)(p<3×10⁻¹⁰ and 6 other genes whose productsparticipate in the UPS system. Of these, 5 genes are involved inubiquitylation, including 2 components of the anaphase promoting complex(APC; ANAPC2, ANAPC4), and one gene is involved in neddylation of thecullins (NEDD8) to promote mitosis.

We next used GeneMANIA²⁰ to build an interaction network relating theMARS set incorporating physical and predicted interactions,co-localization, shared pathways and shared protein domains. 87 genes inthe MARS set formed a single interacting network. Genes that participatein the major functional categories, but are not annotated to have directprotein interactions, were added to this network to produce a corefunctional-interaction module (FIG. 3). Notably, the proteasome and itsassociated proteins constituted a core module linking multiple TGSgenes. Other broad processes that stood out were metabolism, whichincluded genes encoding 2 glycolytic enzymes (GAPDH, PFKL), the G1/Stransition and mitosis steps of the cell cycle and multiple steps inmRNA expression, since the MARS captured multiple DNA binding proteinsexpected to regulate transcription, several RNA polymerases and genesinvolved in mRNA splicing.

Highly Malignant TNBC Epithelial Cells are Selectively Addicted toProteasome Activity

One of the goals of the screen was to point to potential therapeuticapproaches for TNBC. Since the proteasome is a key module in the MARSand proteasome inhibitors are available in the clinic, we firstevaluated the effect of proteasome inhibitors on BPLER and other breastcancer lines.

The proteasome inhibitor bortezomib similarly inhibited proteasomeactivity (FIG. 4A) and induced accumulation of p21 and p27 proteins, twoestablished biological markers of proteasome inhibition, in BPLER andHMLER (FIG. 11A). However, as anticipated, BPLER were more sensitive tobortezomib compared to HMLER (FIG. 4B). Similar results were obtainedusing MG132 and second-generation proteasome inhibitor drugs. Thesensitivity of BPLER cells to bortezomib was specific, since BPLER andHMLER cells responded similarly to treatment with doxorubicin (FIG. 4C).

After low-dose bortezomib treatment, BPLER did not undergo cell cyclearrest and showed clear evidence of DNA fragmentation, caspase-3activation, and PARP1/Mcl-1 cleavage, suggesting that they died ofapoptosis, whereas HMLER underwent G2/M arrest with minimal signs ofapoptosis. Notably, untransformed BPE underwent G2/M arrest and wereresistant to bortezomib-induced apoptosis. Thus, bortezomib was notcytotoxic at this dose (FIG. 4D-4E, and FIG. 11B). This was confirmed byflow cytometry after annexin V and propidium iodide (PI) staining.Moreover, BPLER developed an annexin V+PI+ population that mostlydisappeared after treatment in the presence of the pan-caspase inhibitorzVAD-fmk, confirming the induction of caspase-dependent cell death byproteasome inhibition (FIG. 11C).

Four other epithelial TNBC cells (MB-468, HCC-1143, HCC-1937 and 4T1-E)were also relatively sensitive to bortezomib, whereas ER+(MCF7), HER2+(BT-474) and mesenchymal (MB-231, MB-436) breast cancer cell lines wereresistant, suggesting that TNBC epithelial cell lines are selectivelyaddicted to proteasome activity (FIG. 4F). In the highly malignant 4T1-Ebreast cancer cell line, viable cells that persisted after 18-hourbortezomib treatment neither formed colonies in vitro after 2 weeks, norwere able to initiate tumors in vivo. Similar results were obtained inHCC-1143 in vitro (FIGS. 4G-4H).

Together, these data indicate that TNBC epithelial cells withtumor-initiating potential are exquisitely sensitive to proteasomeinhibition.

Noxa Mediates Bortezomib-Induced Apoptosis in Epithelial TNBC CellLines.

Because the proteasome was so prominent in the MARS and bortezomib wasselectively lethal to highly malignant TNBC epithelial cells, we wantedto understand better the mechanism underlying BPLER and TNBC sensitivityto proteasome inhibition. Toward this goal, we first assessed therelative protein level of key modulators of apoptosis in BPLER and HMLERafter treatment with bortezomib, including regulators of mithocondrialmembrane depolarization and factors acting downstream of mithocondria(FIG. 5A). Because some of these factors, such as Mcl-1, are cleavedupon caspase activation, we performed these experiments in the presenceof zVAD-fmk. In both cell lines, bortezomib induced expression of Mcl-1and the BH3-only proteins Noxa, Bik and Bim, while it inhibitedpost-mithocondrial factors XIAP and c-IAP1. However, bortezomib inducedmithocondrial membrane depolarization in BPLER but not HMLER, indicatingthat pre-mithocondrial factors regulated the selective response of BPLERto proteasome inhibition (FIG. 5B). To identify factors mediatingapoptosis at a functional level, we silenced the expression of 24 genesthat were linked with bortezomib-induced cell death in previous studies.Of these, only Noxa silencing rescued BPLER cell death after bortezomibtreatment to the same extent of the pan-caspase inhibitor zVAD-fmk (FIG.5C). Indeed, Noxa protein is up regulated after treatment withbortezomib and mediates bortezomib-induced apoptosis in BPLER. However,Noxa was not sufficient for bortezomib-induced cell death, since HMLERoverexpressed Noxa after treatment with bortezomib as well.

The accumulation of Noxa in both cells appeared to be regulated bothtranscriptionally and post-transcriptionally, since Noxa proteinincreased at a low bortezomib concentration that showed no change inmRNA (FIGS. 12A-12B). Notably, Noxa mRNA increased ˜21-fold when BPEwere transduced with SV40 early region (BPLE) and did not increasefurther after transduction with activated RAS (BPLER). Furthermore, TNBClines expressed ˜5-25-fold more baseline Noxa mRNA compared tountransformed BPE cells and poorly malignant luminal MCF7 and T47Dcells, suggesting that Noxa transcriptional activation in mammaryepithelial cells occurs upon transformation (FIG. 5D). Accordingly,bortezomib strongly induced Noxa protein in all the TNBC cell linestested, but only weakly in BPE, MCF7 and T47D (FIG. 12C). Thus, TNBCcells are poised to Noxa protein accumulation upon proteasomeinhibition. Of note, Noxa induction upon bortezomib treatment correlatedwith PARP1 cleavage in BPE, luminal MCF7 and epithelial TNBC cell lines(HCC-1143, MB-468 and HCC-1937, FIG. 5E).

Together, these data indicate that Noxa is a key mediator ofbortezomib-induced cell death in TNBC epithelial cells.

Mcl-1 Dependency Underlies TNBC Sensitivity to Proteasome Inhibition.

Since differential expression of Noxa did not explain the selectivesensitivity of BPLER and other epithelial TNBC cell lines to bortezomib,we sought to define the molecular mechanism underlying their selectivedependence upon proteasome inhibition.

Noxa is an established, direct and specific antagonist of Mcl-1, whichstood out as the only bcl-2 family member among BPLER dependency genesin our screen. Thus, we wished to determine whether BPLER selectiveresponse to proteasome inhibition depends on Noxa-mediated inactivationof Mcl-1. To test this, we first assessed Noxa/Mcl1 protein binding uponproteasome and caspase inhibition in BPLER and HMLER. In fact, Noxabound to Mcl-1, but not Bcl-XL, in the 2 cell lines to a similar extent,indicating that Noxa was not just expressed at similar levels, but alsoequally capable of binding Mcl-1 (FIG. 5F).

Then, we assessed BPLER and HMLER dependence upon distinct Bcl-2 familymembers. As expected, Mcl-1 silencing was selectively lethal to BPLERcompared to HMLER, whereas BCL-XL silencing killed both BPLER and HMLERto a similar extent and BCL-2 silencing had no effect in either cellline. Hence, BPLER were selectively and specifically dependent on Mcl-1for survival compared to HMLER, and Mcl-1 silencing was sufficient torecapitulate the lethal effect of proteasome inhibition (FIG. 5G).

In order to validate these findings in other genetic and epigeneticcontexts, we silenced Mcl-1 expression in breast cancer cell lines ofdifferent subtypes. As anticipated, Mcl-1 silencing was lethal toepithelial TNBC cell lines HCC-1937, HCC-1143 and MB-468, but not toluminal MCF7 and mesenchymal MB-231 cells. Notably, MB-468 that wedemonstrated are exquisitely sensitive to bortezomib were alsoexquisitely sensitive to Mcl-1 silencing (FIG. 5H). Thus, other humanepithelial TNBC cell lines, and not just BPLER, are selectivelysensitive to Mcl-1 inhibition.

These data indicate that Mcl-1 dependency underlies the selectiveresponse of TNBC cells to proteasome inhibition. Because epithelial TNBCcells are intrinsically poised to Noxa accumulation and depend on Mcl-1for survival, proteasome inhibition represents a viable therapeuticstrategy to target Mcl-1 dependency in TNBC.

Proteasome Inhibition Suppresses TNBC Outgrowth In Vivo

Our data thus far indicate that highly malignant epithelial TNBC cellsare selectively susceptible to proteasome inhibition. Bortezomib isalready in the clinic for treatment of multiple myeloma and otherlymphomas. To start investigating potential clinical efficacy in TNBC,BPLER tumor-bearing mice were given 1 dose (0.8 mg/kg) of bortezomibintratumorally (i.t.), intraperitoneally (i.p) or intravenously (i.v.)and proteasome activity in the tumor was assessed after 18 hours (FIG.6A). Nearly complete proteasome inhibition was achieved via i.t.injection, whereas i.p. and i.v. routes were less effective. However,increasing the dose of bortezomib to 1.6 mg/kg allowed efficientproteasome activity inhibition via systemic administration (i.v.). Thus,we used biologically active therapeutic regimens (0.8 mg/kg i.t. and 1.6mg/kg i.v.) for subsequent experiments. To assess therapeutic efficacy,we administered bortezomib every 3 days (0.8 mg/kg i.t.) or weekly (1.6mg/kg i.v.) after BPLER tumors, implanted in the flank of Nu/J mice,became palpable. BPLER tumor outgrowth was assessed by tumor volumeduring the treatment period and by tumor weight at the time ofsacrifice. Compared to control mice, tumors in bortezomib-treated micewere on average from 85% (i.t.) to 59% (i.v.) smaller (p<0.01 andp<0.002, respectively), and weighed on average from 91% (i.t) to 63%%(i.v.) less compared to control mice (p<0.001 and p<0.006, respectively,FIG. 5b-e ). Moreover, bortezomib induced Noxa induction and caspase 3cleavage in vivo, as determined by immunohistochemistry and western blotanalysis (FIG. 6F). Thus, bortezomib inhibited the activity of itsintended target, induced Noxa accumulation, triggered apoptosis andsuppressed tumor growth in BPLER tumor xenografts.

To further validate bortezomib efficacy in vivo, we used MB-468 that hadshown dramatic response to proteasome inhibition in vitro. Treatment wasstarted once mice developed palpable tumors. Using either intratumoralor intravenous therapeutic regimens, mice treated with bortezomib for 45days had tumors that were smaller by at least 6-fold compared to controlmice (p<0.0001 in both conditions, FIG. 6G and FIGS. 11A-11C).

Since most of our work was based on cancer cell lines, we sought toassess bortezomib efficacy using primary tumors from TP53 heterozygousBalb/c mice that develop TNBC with a 40-week latency. Breast tumors wereresected from tumor-bearing mice and tumor fragments implanted into themammary fat-pad of TP53^(+/+) Balb/c recipient mice. After implantation,mice were randomized and treated with bortezomib weekly by i.v. infusion(FIG. 6H). Mice treated with bortezomib developed secondary tumors thatwere ˜90% smaller and weighed ˜80% less on average compared to controlmice (p<2.27×10^⁻⁶ and p<0.001, respectively, FIGS. 6I-6J). Histologicalexamination confirmed that these tumors displayed an epithelialphenotype, resembled human TNBC histologically, and stained positive forCK14, thus retaining a TNBC phenotype.

Indeed, bortezomib-based therapy significantly suppressed tumorprogression in a variety of epithelial TNBC models in bothimmunodeficient and immunocompetent mice.

A Chemogenetics Approach Reveals BPLER Selective Sensitivity to HDACInhibitor Drugs.

To identify additional small molecules that might be active againstBPLER, we adopted a “chemogenetic” approach to compare the TGS with theconnectivity map (CMAP), a collection of gene expression profiles ofhuman cancer lines treated with bioactive molecules^(22,23). The CMAPcontains genome-wide mRNA expression data from over 7,000 control vsdrug-treated pairs of cancer cell lines that originated from diversetissues exposed to a subset of 1,309 compounds. We reasoned that smallmolecules that suppress expression of TGS dependency genes might haveselective activity against BPLER. 37 compounds significantlypreferentially suppressed TGS gene expression in the CMAP (Table 5). Weselected 10 drugs from this list for in vitro testing, based partly ontheir potential for clinical use (FIG. 7A). The histone deacetylase(HDAC) inhibitor Trichostatin A (TSA) most significantly suppressed TGSgene expression, which was shown in 182 independent control/TSA-treatedcell experiments (P<0.0001). Treatment with another HDAC inhibitor(Vorinostat) also significantly suppressed expression of the TGS genes.The other drugs examined were Resveratrol, a natural product active inanimal cancer models²⁴; 4,5-dianilinophthalimide, an EGFR inhibitor;L-cavananine, an amino acid analog that competes with L-arginine;Nabumetone, a non-steroidal anti-inflammatory drug; Deferoxamine, aniron chelating agent with anti-cancer properties; Loperamide, anopioid-receptor agonist; Triamterene, a potassium-sparing diuretic; andHesperetin, a bioflavenoid found in citrus fruits. Amongst these, 5small molecules decreased BPLER viability at relatively low doses, ofwhich 2 (the HDAC inhibitors TSA and Vorinostat) were selectively lethalfor BPLER compared to HMLER (FIG. 7B and FIG. 14). Moreover, TSA andvorinostat had no effect on BPE viability at the indicated dose.

The CMAP data predicted that TSA would suppress expression of 52 TGStranscripts by ≧1.8-fold (Table 6). We measured the change in expressionof the top 11 of these transcripts in BPLER and HMLER cells treated withTSA. Ten of these, including Mcl-1 and two proteasome subunit genes(PSMC3, PSMD7), were significantly decreased in BPLER cells treated withTSA (FIG. 7C). All but two of these also had significantly reducedexpression in HMLER cells treated with TSA (FIG. 7D).

TSA synergized with bortezomib and increased apoptosis in BPLER (FIGS.7E-7F), whereas the combination was not cytotoxic in untransformed BPEcells in vitro. Thus, HDAC inhibitor drugs selectively targeted highlymalignant TNBC-initiating cells in the BPLER/HMLER system by regulatingthe expression of multiple genes on which such cells selectively dependfor survival.

Discussion

Here we performed a genome-wide siRNA lethality screen of a geneticallydefined human breast cancer cell line (BPLER) to identifyvulnerabilities and potential drug targets of highly malignant andpoorly differentiated TNBC, the breast cancer subtype with the worstprognosis and response to current treatment. To our knowledge, this isthe first RNAi-based high-throughput functional screen of a clinicallyrelevant breast cancer cell type with a high frequency oftumor-initiating cells. Previous studies have focused on highlymalignant mesenchymal breast tumor-initiating cells that give rise tomesenchymal breast tumors, a rare subtype of TNBC also known as“claudin-low”. BPLER cells represent the first genetically defined modelof tumor-initiating cells that give rise to human triple-negativeepithelial adenocarcinomas, the most common type of TNBC encountered inthe clinic.

Recent studies suggest that TNBC arises from transformed luminalepithelial progenitor cells^(4-6,34,35). Our data lend support to thishypothesis, because oncogenic transformation of human primary BPECepithelial progenitor-like cells generates cancer lines enriched forBT-IC that give rise to tumors closely resembling human TNBC. Thus,BPEC-like epithelial progenitor cells may be the cell of origin of humanTNBCs. This is unlikely to be an artifact of in vitro transformation,because introduction of the same genetic elements intomyoepithelial-like cells (HMEC) does not induce TNBC¹⁰. The oncogenesused to transform BPE cells recapitulate common genetic alterations inTNBC, including loss of function of Tp53³⁶ and Rb³⁷ (which areinactivated by SV40) and activation of RAS signaling³⁸. Indeed,transformation of human primary cells into TNBC tumor-initiating cellsappears to depend on the cell of origin. However, it is possible thatother combinations of oncogenes may also induce TNBC-like phenotypesfrom different cells of origin.

The cancer stem cell (CSC) hypothesis postulates that only a minorsubpopulation of stem-cell-like cancer cells within a tumor retaintumor-initiating potential. While the CSC model may apply to certainlow-grade tumors (including luminal breast tumors), it seems unlikely todescribe TNBC, which are typically poorly differentiated lesions with adiffuse progenitor-like phenotype. The entire population of BPLER cellsuniformly has the phenotype attributed to breast CSCs and only 50 cellsare needed to initiate tumors in immunodeficient mice (we didn't tryinjecting smaller numbers). For poorly differentiated tumors like TNBCthat arise from early progenitor cells, most of the tumor cells arelikely to be tumor-initiating cells. Tumors with an early progenitorphenotype may not rely on malignant stem cells for self-renewal, an ideathat has been supported by data using other poorly differentiatedtumors³⁹. Instead, poorly differentiated transformed bipotent epithelialprogenitors can acquire self-renewing properties, and the bulk of cellsin some poorly differentiated tumors can be capable of tumor initiation.It should be noted that HMLER cells, which do not efficiently generatetumors, also have the phenotype attributed to BT-IC. Thus there is areal need for additional BT-IC markers.

We identified 154 genes, which we termed a malignancy associatedresponse signature (MARS) or TGS, which when silenced are selectivelylethal for BPLER, but not for a closely related cell line (HMLER)derived from the same donor using the same transforming oncogenes. HMLERis myoepithelial-like and is unable to initiate tumors when injected atlow numbers¹⁰. BPLER dependency genes include Mcl-1 and are highlyenriched for proteasome subunits. The proteosome inhibitor bortezomib,currently approved for treatment of multiple myeloma and relapsed mantlecell lymphoma²⁶, causes caspase-dependent apoptosis in TNBC-initiatingepithelial cells. These cells are especially sensitive to proteasomeinhibition, because they selectively depend on Mcl-1 for survival andare intrinsically poised to Noxa protein accumulation, a potentinhibitor of Mcl-1. In fact, a number of human epithelial TNBC celllines, and not just BPLER, appear to be selectively sensitive to Mcl-1inhibition and to proteasome inhibitor drugs that induce Noxa. On theother hand, TNBC cell lines that express Noxa but do not depend on Mcl-1for survival, such as mesenchymal MDA-MB-231, are also resistant toproteasome inhibition. Indeed, proteasome inhibitor drugs can be used totarget Mcl-1 dependency for TNBC therapy. Identifying specific factorsthat regulate Noxa expression as well as Mcl-1 addiction, using themethods and assays described herein, can also provide novel therapeutictargets against TNBC.

Although clinical studies of bortezomib for unselected breast cancersubtypes have not been promising²⁷⁻²⁹, our study indicates that furtherclinical evaluation of proteasome inhibitors for selected TNBC patients,which represent ˜15% of breast cancer patients, is worthwhile. Recentstudies indicate that achieving robust proteasome silencing in solidtumors can represent a major obstacle in the clinic. However,second-generation proteasome inhibitors with better pharmacokineticprofiles or targeting specific components in the ubiquitin-proteasomesystem, currently under clinical development, might hold promise forTNBC therapy. Our data also indicate that combining proteasomeinhibitors with Mcl-1 inhibitors, also in preclinical development, canenhance clinical responses.

In this study we also used a novel approach to identify other potentialdrugs that are active against TNBC. We compared the CMAP that providesdata on changes in global gene expression after exposure to bioactivemolecules for many cancer cell lines with the list of MARS genes toidentify drugs that selectively reduce expression of BPLER dependencygenes. This approach, which illustrates the potential of combining siRNAscreen results with CMAP or similar chemogenetics databases, identifiedHDAC inhibitors as selectively toxic for TNBC-initiating cells in theBPLER/HMLER system. HDAC inhibition suppressed multiple BPLER dependencygenes, including Mcl-1 and proteasome subunits. In fact, TSA andbortezomib had a synergistic effect against BPLER. It is worth notingthat HDAC inhibitors have been previously shown to target multiplemyeloma cells and to synergize with bortezomib. In fact, 2 clinicaltrials for bortezomib/HDAC combination therapy (Velcade/Zolinza) inmultiple myeloma are currently active. Indeed, TNBC-initiating cellsappear to share selective functional dependencies with multiple myelomacells, and indicates the activation of a related oncogenic program indistinct disease entities.

The MARS or TGS biomarkers (or the subset of the 23 biomarkers) can alsobe useful for identifying other potential TNBC drugs. Because the listis of a manageable size, the pathways for which there are multiple hitsor the genes whose products lie at the nodes of the protein-proteininteraction network can be evaluated as drug targets for TNBC, eitherfor conventional drug development or for gene knockdown using small RNAsor antisense oligonucleotides. For example, the MARS also comprisesglycolytic enzyme genes, and glycolysis inhibiting drugs are activelyunder development. We found that BPLER are highly reliant on glycolysisfor ATP generation and are selectively sensitive to glycolysisinhibition.

A recent chemical screen identified the potassium ionophore salinomycinas a candidate drug active against highly malignant mesenchymal HMLERcells knocked down for E-cadherin³⁰. Three hits in our screen, SLC4A5,SCL24A3 and SLC47A1, are potassium-sodium exchange transporters. It isworth investigating whether highly malignant breast cancer cells likeBPLER that more closely resemble human breast tumors are alsoselectively sensitive to inhibition of specific ion transport.

TNBCs are sensitive to drugs like paclitaxel that inhibit mitosis andform the basis of current care. Our screen identified many genesinvolved in mitosis as required for BPLER survival. We verified thatMcl1 protein also declines in BPLER cells arrested in mitosis (bynocodazole, data not shown). Hence, combining proteosome inhibition withdrugs that arrest cells in mitosis can be a synergistic approach.

The hits in our screen involved in the UPS and mitosis (the proteasomeand the APC) overlap with genes identified in a recent genome-wide siRNAscreen of synthetic lethal interactions with KRAS³². Because both BPLERand HMLER are transformed to express constitutively activated RAS, theirdifferential dependence on these hits indicates that the effect of RASactivation is context-dependent and can be especially relevant in BT-ICor poorly differentiated tumors that arise from multipotent progenitorcells. In fact, we previously showed that RAS plays an important role inmaintaining the self-renewal properties of BT-IC³³.

Expression of the 154 member MARS of BPLER dependency genes or thesmaller subset of 23 highly selective hits provides a useful signaturefor, for example, predicting cancer prognosis, such as breast cancerprognosis, response to certain therapeutic drugs or probability ofresistance or metastatic recurrence, as demonstrated herein. Theseresults are also useful for stratifying patients for clinical studies,especially for drugs expected to impact the pathways enriched in thegene set. The prognostic value of this signature can also be used inluminal B subtype tumor prognosis, as we found by retrospective analysisthat it discriminates between good and poor prognosis patients withluminal B subtype tumors.

Methods

Cell Culture.

Human BPE, BPLE, BPLER and HMLER cells were provided by R. Weinberg andT. Ince and grown in WIT medium (Stemgent). All experiments wereperformed with pairs of cells derived from the same patient (BPLER-2 andHMLER-2). Other human cell lines were obtained from ATCC and grown inDMEM, except for MCF7 (MEM) and HCC-1943 and HCC-1937 (RPMI), allsupplemented with 10% FBS unless otherwise indicated. BPLER and HMLERcells were reverse-transfected with 50 nM siRNAs for screening andfunctional experiments using Dharmafect#1 (Dharmacon) or LIPOFECTAMINE2000 (Invitrogen), respectively, in WIT medium. For drug treatment,cells were plated at 50,000-80,000 cells/well in 6-well plates or 1000cells/well in 384-well plates in WIT and treated 24 hr later. Drugtreatment of transfected cells was begun 24 hr after transfection. Cellviability was assessed by CELLTITERGLO (Promega) in 384-well plates orby Trypan-Blue staining in 6-well plates. Chemoluminescence was measuredusing an ENVISION (PerkinElmer) high-throughput plate reader. Cellviability after treatment with BRPA was assessed by Trypan-Bluestaining.

RNA Analysis.

qRT-PCR analysis was performed as described⁴¹. Briefly, total RNA wasextracted with TRIZOL (Invitrogen) and cDNA prepared from 600-900 ngtotal RNA using THERMOSCRIPT RT kit (Invitrogen) as per themanufacturer's instructions. 2.5 ul of diluted cDNA (1:20) was used astemplate for qPCR using POWER SYBR-GREEN MASTER MIX (Applied Biosystems)and BIORAD C1000 THERMAL CYCLER (Biorad). Primer sequences are availableupon request. Relative CT values were normalized to □-actin andconverted to a linear scale using the −ΔCT method.

Protein Analysis.

Immunoblot was performed as described⁴¹. Primary antibodies were asfollows: cleaved Caspase 3 (Cell Signaling, rabbit polyclonal, 1:1000),PARP1 (Santa Cruz, rabbit polyclonal, 1:500), MCL1 (Cell Signaling,Rabbit polyclonal, 1:1000), Bim (Santa Cruz, rabbit polyclonal, 1:500),Bik (Cell Signaling, rabbit polyclonal, 1:1000), Puma (Cell Signaling,rabbit polyclonal, 1:1000), Noxa (Calbiochem, mouse monoclonal, 1:500),Bid (Santa Cruz, rabbit polyclonal, 1:500), Bad (Santa Cruz, rabbitpolyclonal, 1:500). Antibodies were diluted in 5% milk in TBS-T andincubated O.N. at 4° C. Secondary mouse and rabbit HRP-conjugatedantibodies were from Amersham. Protein signal was detected using the ECLPlus kit (Amersham). For flow cytometry, cells were stained aspreviously described³³, using the following fluorescent-conjugatedantibodies: CD44 (BD Biosciences), CD24 (BD Biosciences), CD326 (ESA,Biolegend), Annexin V (Invitrogen). For immunofluorescence microscopy,cells were fixed in 2% PFA for 10 min, permeabilized with 1% Triton X inPBS for 5 min, and incubated with primary antibodies in 0.5% Triton X,1% FBS in PBS for 30 min at RT. Antibodies were: CK18 (Santa Cruz, mousemonoclonal, 1:100) and CK14 (Millipore, mouse monoclonal, 1:100). Afterwashing the cells were stained sequentially withAlexaFluor647-conjugated secondary antibody (Invitrogen, 1:200) and DAPI(Sigma, 1:5000). For immunohistochemistry, BPLER tumor sections wereprepared from paraffin-embedded tissues, deparaffinized and treated with10% citrate buffer for 15 min. Staining was performed using a BIOGENEXIHC DAB kit (Biogenex) following the manufacturer's protocol. Primaryantibodies were: CK14 (Millipore, mouse monoclonal, 1:100), CK5(Millipore, mouse monoclonal, 1:100), HH3 (Cell Signaling, rabbitpolyclonal, 1:100), ER (Abcam, mouse monoclonal, 1:100), Vimentin(Invitrogen, mouse monoclonal 1:100), Ki67 (Millipore, mouse monoclonal,1:100).

In Vivo Experiments.

All animal procedures were performed with IACUC approval. Exponentiallygrowing cells were trypsinized with TRYPLE EXPRESS (Invitrogen),resuspended in a 1:1 WIT-Matrigel solution at the indicated numbers, andinjected subcutaneously in the flank of 4-week old female Nu/J mice(Stock #002019, Jackson Laboratories). For bortezomib treatment,beginning 21 days after BPLER cells were injected (when tumors becamepalpable), mice bearing tumors of comparable size were randomized intotwo groups and treated i.p. q3d with bortezomib 0.5 mg/kg or DMSO,respectively. Mice were sacrificed 16 days after beginning treatment andtumor weight was assessed.

RNAi Screen.

Automated screening procedures were performed at the Harvard ICCB-LScreening Facility using Human siGenome siRNA libraries (Dharmacon).Procedures were optimized and validated for high-throughput screeningunder ICCB-L guidance. Screening conditions were identical for BPLER andHMLER cells. In the primary screen, library plates that contained siRNAsno longer supported by transcript evidence were not screened. For eachcell line, each siRNA or pool of siRNAs was transfected in triplicatewells. Each microplate included 8 negative and 8 positive internalcontrols, which were used to monitor experimental conditions across thescreen. Only microplates with a Z′ factor >0.5 were analyzed (covering98.7% of the siRNA library). WELLMATE rapid plate dispensers andTeflon-coated manifolds (Matrix) were used to dispense the cells. siRNAslibraries were transferred into 384-well assay plates using liquidhandling robots (VELOCITY 11 BRAVO). siRNAs were reverse-transfected ata final concentration of 50 nM in 384-well white/clear microplates(Corning Cat. #3707) using Dharmafect #1 (Dharmacon) in WIT medium.Fresh medium was added after 24 hours and cell viability was assessed byCELLTITERGLO (Promega) 3 days after transfection using an ENVISIONhigh-throughput plate reader (Perkin-Elmer).

Screening Hit Selection.

Viability scores were judged based on a combination of parameters,including relative standard deviation among replicates (RSD), medianabsolute deviation (MAD)-based Z score (Z score), fold-change from theplate median (FC), and BPLER/HMLER FC ratio (R)⁴². For each set oftriplicate plates, siRNAs with RSD >0.25 were excluded. Any siRNAscausing severe cytotoxicity to HMLER(HMLER Z score ≦−3 or HMLER FC ≦0.5)were also excluded from further analysis. siRNAs were considered hits ifthey satisfied the following criteria: BPLER Z score ≦−1.5, BPLER FC≦0.75, R≦0.75. Positive hits were classified into highly, moderately ormodestly selective based on R values. The positive hits were subjectedto a secondary screen in which cells were transfected individually withthe 4 siRNAs in the positive pools. Hits were considered validated ifwells treated with at least one siRNA in the pool met the same criteriaused in the primary screen.

Microarray Analysis.

Human breast tumor array data was obtained from two previous studies onAgilent and Affymetrix arrays (UNC dataset¹², 337 cases and Richardsondataset¹³, 47 cases). The gene expression profile of BPLER tumorexplants was acquired using Affymetrix arrays. For cross platformcomparison arrays were rank normalized using only genes common to allarrays. To account for the fact that all of the stromal genes in theBPLER xenografts are of mouse origin, a principal component analysis wasused to identify the major component that separated the tumor and BPLERarrays and these genes were excluded from the subsequent analysis. Thesamples were co-clustered based on the remaining genes that weredifferentially expressed across the tumor dataset by Ward's minimumvariance method according to Spearman correlation coefficients (GEOreferences GSE18229, GSE3744 and GSE6885).

Survival Analysis of Primary Tumor Datasets.

The expression of the TGS and highly specific gene signatures wereassessed in each tumor sample in the NKI dataset¹⁴ using thesingle-sample GSEA enrichment score, as previously described⁴³. Briefly,each sample was rank-normalized and an enrichment score was obtained bya weighted sum of the difference between the empirical distributionfunctions of the genes in the signature and that of the remaining genes.The enrichment scores were classified as low and high expression,according to the p-value associated with up-regulation of expression ofTGS genes. Tumors whose score ranked with p<0.1 were assigned to thehigh expression group and the remaining tumors were defined as low.Survival and metastasis-free survival curves were derived using theKaplan-Meier method.

Tables for Examples 1 and 2

TABLE 1 Genes that Decreased BPLER Viability BPLER/ Entrez Gene MAD MADFold Fold HMLER ID Accession # Gene Symbol BPLER HMLER BPLER HMLER Ratio79864 NM_024806 C11orf63 −1.69 −2.37 0.709 0.63 1.12 54934 NM_017822C12orf41 −1.53 −1.15 0.652 0.84 0.78 80209 NM_025138 C13orf23 −1.78−1.61 0.680 0.72 0.95 221150 NM_145061 C13orf3 −1.66 −0.47 0.694 0.920.76 283598 NM_182560 C14orf177 −2.78 −2.57 0.401 0.48 0.83 283635NM_173607 C14orf24 −1.66 −1.94 0.638 0.60 1.06 64207 NM_024496 C14orf4−1.63 −0.21 0.731 0.97 0.75 348110 NM_182616 C15orf38 −2.27 −2.24 0.6260.67 0.93 90381 NM_152259 C15orf42 −1.66 −1.47 0.666 0.78 0.85 283897NM_175900 C16orf54 −1.80 −1.98 0.611 0.60 1.02 146850 XM_375404 C17orf38−2.01 −1.40 0.643 0.79 0.82 79018 NM_024052 C17orf39 −2.16 −3.69 0.6460.42 1.53 79002 NM_024038 C19orf43 −2.54 −2.67 0.587 0.59 0.99 79098NM_023938 C1orf116 −1.83 −1.56 0.695 0.75 0.92 26099 NM_015609 C1orf144−2.02 −1.72 0.680 0.71 0.95 149466 NM_182517 C1orf210 −1.51 −1.60 0.7310.76 0.96 54823 NM_017673 C1orf26 −1.93 −1.87 0.677 0.74 0.91 79078NM_024097 C1orf50 −1.59 −1.40 0.741 0.78 0.96 79871 NM_024813 C1orf82−3.98 −3.04 0.313 0.53 0.59 140701 NM_080622 C20orf135 −1.83 −2.55 0.6760.63 1.08 253143 NM_173566 C22orf30 −1.73 −0.57 0.728 0.90 0.81 56947NM_020194 C2orf33 −1.84 −1.21 0.703 0.81 0.86 80304 NM_025203 C2orf44−1.89 −1.33 0.665 0.75 0.88 57415 NM_020685 C3orf14 −1.71 −1.56 0.7210.76 0.95 25871 NM_015412 C3orf17 −1.78 −1.18 0.721 0.82 0.88 131831NM_152394 C3orf44 −1.58 −1.76 0.715 0.72 0.99 206412 XM_116497 C6orf163−1.72 −1.54 0.728 0.73 1.00 253714 NM_198468 C6orf167 −2.12 −1.24 0.6650.79 0.84 221261 XM_168053 C6orf184 −2.22 −1.47 0.650 0.75 0.86 221718NM_152738 C6orf218 −1.84 −1.20 0.709 0.79 0.90 84302 NM_032342 C9orf125−2.46 −2.37 0.528 0.61 0.87 138240 XM_059954 C9orf57 −1.71 −1.30 0.6680.77 0.86 55684 NM_024718 C9orf86 −1.61 −1.92 0.654 0.71 0.92 203245NM_197956 C9orf90 −1.59 −1.10 0.749 0.82 0.91 56344 NM_019855 CABP5−1.81 −0.50 0.708 0.92 0.77 773 NM_000068 CACNA1A −2.11 −1.83 0.607 0.680.90 775 NM_000719 CACNA1C −1.67 −1.39 0.688 0.76 0.91 83698 NM_031468CALN1 −1.61 −0.95 0.695 0.84 0.83 55450 NM_018584 CAMK2N1 −3.80 −1.540.626 0.72 0.87 824 NM_001748 CAPN2 −2.18 −2.55 0.651 0.61 1.08 84290NM_032330 CAPNS2 −1.68 −1.12 0.681 0.81 0.84 84433 NM_032415 CARD11−1.64 −0.70 0.732 0.88 0.83 64170 NM_022352 CARD9 −1.53 −0.81 0.744 0.870.86 57524 NM_020764 CASKIN1 −1.84 −0.86 0.701 0.87 0.81 9994 NM_012115CASP8AP2 −2.05 −1.66 0.631 0.70 0.90 863 NM_005187 CBFA2T3 −2.46 −1.320.673 0.74 0.90 79872 NM_024814 CBLL1 −2.41 −3.24 0.585 0.50 1.17 56267NM_019610 CCBL2 −1.88 −1.17 0.695 0.82 0.85 80125 NM_182791 CCDC33 −1.80−0.89 0.675 0.84 0.80 120935 NM_182496 CCDC38 −2.30 −2.54 0.588 0.650.91 152185 NM_144718 CCDC52 −1.96 −1.89 0.699 0.70 0.99 29080 NM_014167CCDC59 −1.68 −1.50 0.702 0.77 0.92 83987 NM_032040 CCDC8 −1.89 −1.820.643 0.70 0.91 283234 NM_032251 CCDC88B −1.73 −0.89 0.638 0.83 0.7754908 NM_017785 CCDC99 −2.46 −3.48 0.420 0.48 0.88 6361 NM_002987 CCL17−2.11 −2.30 0.610 0.64 0.96 6367 NM_002990 CCL22 −2.48 −1.42 0.685 0.790.87 6370 NM_005624 CCL25 −1.69 −1.03 0.688 0.83 0.83 10344 NM_006072CCL26 −2.37 −1.16 0.697 0.82 0.85 891 NM_031966 CCNB1 −1.92 −1.21 0.6520.78 0.83 8812 NM_003858 CCNK −3.07 −2.54 0.443 0.54 0.81 1235 NM_004367CCR6 −1.77 −1.74 0.674 0.73 0.92 1237 NM_005201 CCR8 −1.61 −0.87 0.7040.86 0.82 23552 NM_012119 CCRK −1.75 −1.15 0.744 0.84 0.88 10575NM_006430 CCT4 −2.55 −1.70 0.538 0.69 0.78 930 NM_001770 CD19 −2.12−0.99 0.726 0.85 0.85 910 NM_001764 CD1B −1.90 −1.02 0.657 0.82 0.804345 NM_005944 CD200 −1.53 −1.77 0.706 0.71 1.00 934 NM_013230 CD24−2.59 −2.38 0.527 0.57 0.92 51744 NM_016382 CD244 −1.51 −1.13 0.725 0.800.91 10849 NM_012099 CD3EAP −1.76 −0.24 0.746 0.96 0.78 962 NM_001778CD48 −1.59 −1.42 0.657 0.80 0.83 55143 NM_018101 CDCA8 −2.19 −3.49 0.4800.48 1.00 8558 NM_003674 CDK10 −1.81 −1.03 0.734 0.86 0.86 80279NM_025197 CDK5RAP3 −3.10 −2.74 0.548 0.63 0.87 125931 NM_198444 CEACAM20−3.05 −2.95 0.451 0.59 0.77 1951 NM_001407 CELSR3 −1.53 −0.80 0.718 0.870.83 1062 NM_001813 CENPE −2.75 −2.61 0.590 0.58 1.03 2491 NM_006733CENPI −1.54 −1.59 0.692 0.76 0.92 79172 NM_024322 CENPO −2.42 −2.220.597 0.65 0.92 22897 XM_374936 CEP164 −3.04 −2.08 0.565 0.66 0.86 1073NM_021914 CFL2 −1.80 −1.17 0.614 0.81 0.75 114335 NM_033377 CGB1 −1.57−1.01 0.716 0.82 0.87 79094 NM_024111 CHAC1 −2.41 −2.44 0.602 0.61 0.981108 NM_001273 CHD4 −2.00 −2.22 0.702 0.64 1.10 1111 NM_001274 CHEK1−4.19 −3.53 0.387 0.52 0.74 27243 NM_014453 CHMP2A −1.95 −3.58 0.6550.45 1.47 91782 NM_152272 CHMP7 −1.70 −2.32 0.676 0.67 1.01 91851NM_145234 CHRDL1 −1.64 −1.11 0.689 0.84 0.82 1131 NM_000740 CHRM3 −1.58−2.09 0.709 0.68 1.05 8973 NM_004198 CHRNA6 −2.24 −0.51 0.722 0.92 0.781178 NM_001828 CLC −1.52 −0.67 0.670 0.89 0.75 1186 NM_001287 CLCN7−2.32 −1.62 0.716 0.73 0.98 137075 NM_194284 CLDN23 −1.81 −1.03 0.6550.83 0.79 7123 NM_003278 CLEC3B −2.02 −1.63 0.712 0.76 0.93 25932NM_013943 CLIC4 −2.44 −1.65 0.699 0.73 0.96 55907 NM_018686 CMAS −1.63−1.53 0.637 0.78 0.82 1259 NM_000087 CNGA1 −2.20 −1.70 0.604 0.70 0.8710256 NM_006314 CNKSR1 −2.74 −3.15 0.599 0.57 1.05 23019 NM_016284 CNOT1−2.74 −2.06 0.571 0.67 0.85 1271 NM_001842 CNTFR −2.00 −0.63 0.722 0.900.80 53942 NM_014361 CNTN5 −1.72 −1.95 0.689 0.65 1.06 1297 NM_001851COL9A1 −3.17 −2.16 0.605 0.66 0.91 150684 NM_152516 COMMD1 −2.28 −0.950.648 0.85 0.76 9276 NM_004766 COPB2 −4.48 −3.75 0.343 0.49 0.70 22820NM_016128 COPG −3.44 −2.50 0.506 0.59 0.86 8533 NM_003653 COPS3 −1.63−0.62 0.705 0.89 0.79 10987 NM_006837 COPS5 −1.51 −1.19 0.727 0.79 0.9210980 NM_006833 COPS6 −2.32 −1.14 0.666 0.81 0.82 22818 NM_016057 COPZ1−3.63 −2.53 0.479 0.58 0.82 1325 NM_001302 CORT −1.63 −2.84 0.667 0.551.22 1325 NM_001302 CORT −2.15 −3.10 0.726 0.53 1.36 64506 NM_030594CPEB1 −1.60 −1.93 0.743 0.70 1.06 1369 NM_001308 CPN1 −1.87 −0.86 0.7010.87 0.81 1370 J05158 CPN2 −1.61 −1.29 0.705 0.77 0.92 58487 NM_021212CREBZF −2.50 −1.40 0.668 0.73 0.92 9282 NM_004229 CRSP2 −2.48 −1.880.690 0.71 0.98 9441 NM_004831 CRSP7 −3.43 −2.36 0.570 0.63 0.90 1436NM_005211 CSF1R −1.85 −2.13 0.730 0.71 1.03 1446 NM_001890 CSN1S1 −1.61−2.03 0.646 0.68 0.95 1459 NM_001896 CSNK2A2 −2.56 −2.32 0.627 0.68 0.9280777 NM_030579 CYB5B −1.68 −1.91 0.691 0.66 1.05 1545 NM_000104 CYP1B1−2.01 −0.30 0.724 0.95 0.76 1595 NM_000786 CYP51A1 −1.66 −2.44 0.6510.61 1.06 153090 NM_032552 DAB2IP −3.29 −2.88 0.491 0.55 0.89 23142NM_015115 DCUN1D4 −1.71 −0.53 0.726 0.92 0.79 245932 NM_153289 DEFB119−1.78 −1.33 0.721 0.77 0.93 140850 NM_139074 DEFB127 −2.20 −1.40 0.6090.79 0.77 1676 NM_004401 DFFA −2.36 −1.36 0.620 0.79 0.78 10901NM_021004 DHRS4 −2.40 −1.05 0.653 0.83 0.79 79665 NM_024612 DHX40 −3.28−3.35 0.433 0.48 0.90 27122 NM_013253 DKK3 −2.46 −2.37 0.565 0.63 0.891750 XM_376652 DLX6 −2.60 −2.26 0.655 0.56 1.17 29958 NM_013391 DMGDH−3.45 −2.47 0.661 0.55 1.20 1763 XM_166103 DNA2L −2.54 −2.55 0.463 0.590.78 1788 NM_022552 DNMT3A −1.70 −2.24 0.728 0.65 1.11 116092 NM_052951DNTTIP1 −1.71 −1.22 0.692 0.78 0.89 220164 NM_152721 DOK6 −2.81 −1.660.556 0.72 0.78 29980 NM_145794 DONSON −4.14 −3.46 0.268 0.46 0.58 1802NM_001384 DPH2 −1.91 −1.63 0.589 0.74 0.80 10231 NM_005822 DSCR1L1 −1.52−1.97 0.632 0.68 0.93 1837 NM_001390 DTNA −1.52 −1.54 0.667 0.77 0.87151636 NM_138287 DTX3L −1.89 −1.78 0.707 0.72 0.98 64118 NM_022156 DUS1L−2.02 −0.85 0.662 0.86 0.77 1852 NM_001395 DUSP9 −2.13 −1.70 0.685 0.720.95 143241 NM_138812 DYDC1 −1.82 −1.12 0.676 0.83 0.82 84332 NM_032372DYDC2 −1.66 −0.96 0.687 0.84 0.81 1781 NM_001378 DYNC1I2 −2.06 −1.720.732 0.74 0.98 1894 NM_018098 ECT2 −2.13 −0.93 0.733 0.85 0.86 1915NM_001402 EEF1A1 −2.11 −1.76 0.620 0.69 0.90 29904 NM_013302 EEF2K −2.93−2.83 0.570 0.61 0.93 60678 NM_021937 EEFSEC −1.98 −0.93 0.666 0.84 0.79283229 NM_173584 EFCAB4A −2.38 −2.66 0.494 0.47 1.05 65989 NM_023932EGFL9 −1.74 −0.45 0.715 0.93 0.77 112399 NM_022073 EGLN3 −1.61 −1.820.678 0.72 0.94 8668 NM_003757 EIF3S2 −2.76 −1.95 0.729 0.65 1.13 63036NM_033440 ELA2A −1.51 −0.80 0.745 0.87 0.86 1996 NM_021952 ELAVL4 −2.31−1.77 0.583 0.68 0.85 1999 NM_004433 ELF3 −1.90 −1.53 0.749 0.70 1.07256364 NM_153265 EML3 −1.86 −2.27 0.621 0.54 1.16 2047 NM_004441 EPHB1−2.11 −1.45 0.688 0.80 0.86 2066 NM_005235 ERBB4 −1.74 −2.36 0.743 0.681.10 2077 NM_006494 ERF −2.70 −1.18 0.641 0.77 0.83 90332 NM_138568EXOC3L2 −2.04 −1.45 0.591 0.79 0.75 5394 NM_002685 EXOSC10 −2.43 −2.010.518 0.67 0.77 3995 NM_021727 FADS3 −1.73 −1.79 0.653 0.73 0.90 11170NM_007177 FAM107A −2.39 −1.05 0.660 0.83 0.80 26071 NM_015582 FAM127B−2.09 −2.47 0.670 0.60 1.11 80097 NM_025029 FAM128B −1.75 −1.31 0.6800.77 0.88 9715 NM_014690 FAM131B −1.82 −3.96 0.599 0.28 2.17 83982NM_032036 FAM14A −1.53 −0.98 0.712 0.83 0.86 54801 NM_017645 FAM29A−2.20 −1.54 0.633 0.79 0.80 26240 NM_012135 FAM50B −2.43 −1.62 0.6180.73 0.84 286336 NM_033387 FAM78A −1.82 −1.22 0.668 0.82 0.81 84985NM_032899 FAM83A −3.11 −2.64 0.490 0.52 0.94 2176 NM_000136 FANCC −1.61−1.61 0.709 0.71 1.00 2178 NM_021922 FANCE −2.19 −2.26 0.600 0.59 1.012197 NM_001997 FAU −3.01 −1.72 0.641 0.72 0.89 54850 NM_017703 FBXL12−1.60 −0.42 0.748 0.94 0.80 283807 NM_203373 FBXL22 −1.74 −0.84 0.6400.82 0.78 93611 NM_033182 FBXO44 −2.30 −2.46 0.570 0.65 0.88 26270NM_018438 FBXO6 −2.11 −1.31 0.688 0.79 0.87 6468 NM_022039 FBXW4 −2.27−0.91 0.661 0.85 0.78 2213 NM_004001 FCGR2B −2.14 −1.74 0.724 0.74 0.982222 NM_004462 FDFT1 −2.11 −1.52 0.663 0.77 0.86 2865 NM_005304 FFAR3−1.56 −0.97 0.714 0.85 0.84 2268 NM_005248 FGR −2.67 −2.20 0.610 0.700.87 84929 NM_032843 FIBCD1 −2.44 −1.47 0.598 0.73 0.82 80099 NM_025031FLJ21075 −2.44 −2.10 0.553 0.63 0.88 80154 NM_025084 FLJ22795 −2.79−2.57 0.493 0.54 0.91 220042 NM_145018 FLJ25416 −2.57 −2.05 0.594 0.650.91 254048 XM_376679 FLJ25778 −2.90 −2.67 0.543 0.55 0.99 152756NM_153027 FLJ31659 −2.26 −1.93 0.652 0.70 0.93 201283 NM_153032 FLJ32065−2.17 −1.88 0.657 0.69 0.96 400629 NM_207459 FLJ35767 −2.91 −2.91 0.4580.49 0.94 168455 NM_175884 FLJ36031 −2.00 −1.95 0.653 0.69 0.94 151258NM_173512 FLJ39822 −1.66 −0.46 0.744 0.93 0.80 147699 NM_178494 FLJ40125−1.65 −1.34 0.708 0.80 0.88 206338 NM_173800 FLJ90650 −1.90 −0.72 0.7020.87 0.81 2324 NM_002020 FLT4 −1.85 −0.86 0.729 0.88 0.83 8061 NM_005438FOSL1 −2.05 −0.85 0.727 0.83 0.87 2300 NM_005250 FOXL1 −2.14 −1.86 0.7160.63 1.13 116113 NM_138457 FOXP4 −1.63 −1.72 0.693 0.75 0.92 53826NM_022003 FXYD6 −1.58 −2.08 0.742 0.71 1.04 2585 NM_002044 GALK2 −2.75−2.39 0.598 0.67 0.89 253959 XM_210022 GARNL1 −1.67 −1.34 0.737 0.770.95 2618 NM_000819 GART −2.28 −1.56 0.713 0.69 1.03 2633 NM_002053 GBP1−2.12 −3.02 0.711 0.52 1.36 2657 NM_001492 GDF1 −1.98 −2.39 0.748 0.641.17 124975 NM_153338 GGT6 −1.99 −2.23 0.647 0.69 0.94 168537 NM_153236GIMAP7 −2.20 −1.13 0.620 0.82 0.76 256356 NM_152776 GK5 −2.15 −1.510.546 0.69 0.79 83468 NM_031302 GLT8D2 −2.03 −1.69 0.616 0.72 0.85 10691NM_006582 GMEB1 −2.09 −1.57 0.723 0.69 1.04 94235 NM_033258 GNG8 −1.55−1.78 0.689 0.73 0.94 27238 NM_015698 GPKOW −1.68 −0.44 0.700 0.93 0.75283554 XM_290615 GPR137C −2.18 −1.55 0.513 0.68 0.75 390212 NM_206997GPR152 −1.97 −1.51 0.627 0.73 0.86 2846 NM_005296 GPR23 −1.73 −1.570.682 0.75 0.91 2852 NM_001505 GPR30 −1.85 −1.10 0.661 0.82 0.80 2853NM_005299 GPR31 −1.71 −1.04 0.687 0.84 0.82 2854 NM_001506 GPR32 −1.77−2.67 0.675 0.59 1.14 2866 NM_005305 GPR42 −1.69 −0.86 0.690 0.87 0.8010149 NM_005756 GPR64 −1.60 −1.69 0.705 0.74 0.95 53831 NM_020370 GPR84−2.09 −1.84 0.615 0.71 0.86 53836 NM_023915 GPR87 −1.90 −2.10 0.653 0.670.98 285513 NM_198281 GPRIN3 −1.62 −2.88 0.705 0.58 1.21 80852 XM_042936GRIP2 −2.15 −2.41 0.610 0.57 1.08 2926 NM_002092 GRSF1 −1.83 −1.95 0.6040.71 0.85 84163 NM_173537 GTF2IRD2 −1.55 −1.00 0.669 0.85 0.79 26164NM_015666 GTPBP5 −2.03 −1.11 0.681 0.82 0.83 2978 NM_000409 GUCA1A −1.51−1.53 0.704 0.75 0.94 3000 NM_000180 GUCY2D −2.59 −1.81 0.620 0.75 0.8260484 NM_021817 HAPLN2 −1.85 −1.02 0.687 0.83 0.83 253018 NM_181717HCG27 −2.47 −1.40 0.611 0.76 0.80 57493 XM_087386 HEG1 −2.41 −1.43 0.6100.78 0.78 3280 NM_005524 HES1 −1.52 −1.64 0.673 0.75 0.90 57801NM_021170 HES4 −2.53 −2.94 0.455 0.57 0.80 135114 NM_138571 HINT3 −1.60−1.56 0.675 0.74 0.91 3093 NM_005339 HIP2 −2.23 −2.50 0.711 0.63 1.138479 NM_003609 HIRIP3 −1.76 −1.71 0.665 0.72 0.92 23262 NM_015216HISPPD1 −2.01 −0.62 0.744 0.88 0.85 121504 NM_175054 HIST4H4 −2.04 −2.200.640 0.69 0.93 3099 NM_000189 HK2 −2.19 −1.49 0.678 0.80 0.85 3146NM_002128 HMGB1 −1.91 −1.64 0.722 0.77 0.94 3156 NM_000859 HMGCR −2.02−1.24 0.743 0.75 0.99 3150 NM_004965 HMGN1 −1.80 −1.16 0.654 0.81 0.81220988 NM_194247 HNRPA3 −2.18 −2.51 0.656 0.58 1.14 50863 NM_016522 HNT−1.81 −2.15 0.611 0.69 0.89 84525 NM_032495 HOP −1.53 −0.26 0.748 0.960.78 3238 NM_021193 HOXD12 −2.65 −2.00 0.675 0.62 1.09 154791 NM_197964HSPC268 −3.61 −2.71 0.441 0.57 0.78 23553 NM_012269 HYAL4 −3.54 −2.990.444 0.52 0.86 3382 NM_004968 ICA1 −1.70 −1.41 0.659 0.78 0.84 23463NM_012405 ICMT −1.81 −0.73 0.708 0.88 0.81 3467 NM_002177 IFNW1 −2.83−1.85 0.636 0.72 0.88 3543 NM_020070 IGLL1 −1.72 −0.92 0.666 0.85 0.783550 NM_006083 IK −4.13 −3.31 0.469 0.50 0.93 3596 NM_002188 IL13 −1.72−1.81 0.666 0.71 0.94 3601 NM_002189 IL15RA −2.13 −2.03 0.725 0.69 1.053553 NM_000576 IL1B −2.98 −2.49 0.617 0.62 0.99 3611 NM_004517 ILK −2.18−1.69 0.568 0.73 0.77 55364 NM_018439 IMPACT −1.68 −2.16 0.634 0.68 0.933619 NM_020238 INCENP −3.05 −3.25 0.386 0.51 0.76 26173 XM_291222 INTS1−1.88 −1.39 0.704 0.77 0.91 57508 NM_020748 INTS2 −2.14 −0.99 0.654 0.850.77 79711 NM_024658 IPO4 −1.53 −1.42 0.732 0.78 0.94 79192 XM_380171IRX1 −2.16 −1.71 0.628 0.74 0.85 122961 NM_194279 ISCA2 −1.58 −1.790.719 0.75 0.95 23479 NM_014301 ISCU −2.62 −1.60 0.589 0.74 0.79 3713NM_005547 IVL −1.73 −1.61 0.653 0.75 0.87 126306 NM_144616 JSRP1 −1.72−2.33 0.690 0.67 1.02 353219 NM_181337 KAAG1 −1.55 −1.04 0.709 0.81 0.87126823 NM_152366 KARCA1 −1.57 −0.86 0.718 0.88 0.81 3780 NM_002248 KCNN1−1.64 −0.72 0.668 0.89 0.75 3785 NM_004518 KCNQ2 −2.29 −1.30 0.734 0.790.93 9132 NM_004700 KCNQ4 −2.43 −1.89 0.709 0.69 1.03 283518 NM_173605KCNRG −1.82 −1.50 0.621 0.70 0.88 57582 XM_029962 KCNT1 −2.28 −2.420.726 0.60 1.21 202559 NM_152688 KHDRBS2 −2.54 −2.08 0.601 0.64 0.9323334 NM_015284 KIAA0467 −1.60 −0.88 0.740 0.86 0.86 23354 XM_049237KIAA0841 −2.55 −1.54 0.595 0.75 0.79 23383 XM_048457 KIAA0892 −1.62−0.89 0.739 0.86 0.86 23379 XM_029101 KIAA0947 −2.87 −2.25 0.545 0.640.85 23325 NM_015275 KIAA1033 −1.95 −0.59 0.690 0.91 0.76 57179NM_020444 KIAA1191 −1.99 −1.20 0.676 0.82 0.83 57535 NM_020775 KIAA1324−2.62 −1.57 0.574 0.76 0.76 57650 NM_020890 KIAA1524 −1.65 −0.95 0.7220.84 0.86 80256 NM_025182 KIAA1539 −1.78 −1.41 0.677 0.75 0.90 57703XM_034594 KIAA1604 −2.57 −2.89 0.566 0.51 1.12 165215 NM_177454 KIAA1946−1.61 −1.30 0.719 0.80 0.90 3832 NM_004523 KIF11 −4.90 −3.56 0.267 0.420.64 113220 NM_138424 KIF12 −3.83 −2.76 0.429 0.55 0.78 81930 NM_031217KIF18A −3.12 −2.18 0.532 0.64 0.82 9493 NM_004856 KIF23 −3.15 −2.680.530 0.57 0.94 57565 NM_020805 KLHL14 −1.71 −1.11 0.710 0.81 0.88 57563NM_020803 KLHL8 −2.16 −1.73 0.636 0.71 0.90 25818 NM_012427 KLK5 −2.11−2.03 0.669 0.68 0.99 11202 NM_007196 KLK8 −2.18 −0.72 0.689 0.88 0.793837 NM_002265 KPNB1 −3.32 −2.97 0.384 0.47 0.81 3887 NM_002281 KRT81−2.14 −2.40 0.587 0.61 0.96 3857 NM_000226 KRT9 −1.68 −1.55 0.672 0.750.90 81851 NM_030967 KRTAP1-1 −1.73 −1.62 0.683 0.71 0.96 337959NM_181621 KRTAP13-2 −2.33 −2.54 0.615 0.63 0.98 3916 NM_005561 LAMP1−1.60 −1.47 0.678 0.78 0.87 353139 NM_178428 LCE2A −1.82 −1.26 0.6590.77 0.86 79143 NM_024298 LENG4 −2.71 −2.73 0.475 0.56 0.85 114783XM_055866 LMTK3 −1.79 −0.97 0.646 0.84 0.77 146325 NM_145270 LOC146325−1.84 −1.93 0.673 0.71 0.94 147646 XM_085833 LOC147646 −1.50 −1.24 0.7340.82 0.89 150223 XM_097886 LOC150223 −2.43 −1.33 0.624 0.79 0.79 161247NM_203402 LOC161247 −2.02 −1.04 0.688 0.84 0.82 196264 NM_198275LOC196264 −1.62 −2.03 0.720 0.68 1.06 283152 XM_378314 LOC283152 −1.83−2.30 0.591 0.53 1.11 283547 XM_378454 LOC283547 −2.22 −1.59 0.507 0.670.76 283871 XM_208887 LOC283871 −1.52 −1.01 0.666 0.79 0.84 339745NM_001001664 LOC339745 −1.63 −3.03 0.726 0.55 1.31 374973 XM_371248LOC374973 −2.30 −1.67 0.572 0.70 0.82 399947 NM_207645 LOC399947 −1.73−2.10 0.676 0.63 1.07 400451 NM_207446 LOC400451 −1.79 −2.58 0.665 0.551.22 90120 XM_379680 LOC90120 −1.53 −0.65 0.693 0.91 0.76 4023 NM_000237LPL −1.88 −2.27 0.742 0.64 1.15 84918 NM_032832 LRP11 −1.57 −1.14 0.7030.80 0.88 10128 NM_133259 LRPPRC −1.84 −1.10 0.734 0.85 0.87 9684NM_014665 LRRC14 −1.59 −1.60 0.694 0.74 0.94 55631 NM_017768 LRRC40−1.85 −1.37 0.642 0.78 0.82 115353 NM_052940 LRRC42 −1.82 −0.93 0.6610.87 0.76 116064 XM_057296 LRRC58 −2.54 −3.12 0.516 0.55 0.93 65999NM_023942 LRRC61 −1.88 −1.97 0.690 0.69 1.00 51599 NM_015925 LSR −2.61−2.86 0.501 0.53 0.94 55692 NM_018032 LUC7L −1.61 −1.34 0.644 0.81 0.8066004 NM_177477 LYNX1 −2.42 −2.71 0.597 0.58 1.04 116068 XM_371760LYSMD3 −2.17 −1.99 0.583 0.71 0.82 4101 NM_005361 MAGEA2 −2.02 −2.230.593 0.66 0.90 9175 NM_004721 MAP3K13 −1.50 −0.83 0.697 0.87 0.80 5594NM_002745 MAPK1 −1.57 −0.52 0.683 0.91 0.75 225689 NM_139021 MAPK15−1.74 −0.67 0.744 0.91 0.82 23031 XM_038150 MAST3 −1.53 −1.52 0.705 0.750.93 4147 NM_002380 MATN2 −1.79 −2.39 0.639 0.64 1.00 4149 NM_002382 MAX−1.53 −1.29 0.700 0.79 0.88 4152 NM_002384 MBD1 −2.24 −0.88 0.725 0.830.87 125997 NM_144614 MBD3L2 −1.88 −1.67 0.664 0.77 0.86 4199 NM_002395ME1 −2.62 −1.81 0.669 0.64 1.05 4200 NM_002396 ME2 −1.52 −0.99 0.6940.85 0.82 4201 NM_014623 MEA1 −1.59 −1.92 0.679 0.71 0.96 84246NM_032286 MED10 −1.54 −1.68 0.708 0.73 0.97 80306 NM_025205 MED28 −2.77−2.63 0.497 0.53 0.94 10001 NM_005466 MED6 −2.01 −1.99 0.748 0.69 1.08112950 NM_052877 MED8 −2.06 −2.20 0.602 0.64 0.93 1953 XM_031401 MEGF6−2.16 −1.92 0.528 0.69 0.76 4225 NM_005925 MEP1B −2.09 −1.54 0.600 0.750.80 59274 NM_022566 MESDC1 −1.81 −0.93 0.653 0.85 0.77 23184 XM_370880MESDC2 −2.08 −1.53 0.602 0.75 0.81 4236 NM_005926 MFAP1 −2.75 −3.080.447 0.53 0.84 84310 NM_032350 MGC11257 −1.65 −1.37 0.688 0.77 0.8994107 NM_053045 MGC14327 −2.77 −3.16 0.471 0.55 0.86 84848 NM_032762MGC16121 −1.64 −1.11 0.732 0.80 0.92 93624 XM_291105 MGC21874 −1.95−2.84 0.628 0.59 1.07 200424 XM_371501 MGC22014 −1.61 −1.03 0.721 0.830.86 84730 NM_032644 MGC2452 −1.56 −1.67 0.746 0.70 1.07 169166NM_152628 MGC39715 −1.68 −1.66 0.712 0.74 0.96 84752 NM_033309 MGC4655−2.75 −1.72 0.548 0.69 0.80 202915 NM_152689 MGC9712 −2.69 −1.98 0.5750.67 0.86 2872 NM_017572 MKNK2 −1.89 −1.03 0.635 0.84 0.76 64976NM_003776 MRPL40 −1.77 −0.98 0.711 0.84 0.84 122704 NM_181307 MRPL52−2.03 −1.61 0.636 0.78 0.82 65005 NM_031420 MRPL9 −1.50 −0.13 0.747 0.970.77 64951 NM_032014 MRPS24 −1.72 −0.67 0.712 0.89 0.80 10903 NM_181873MTMR11 −2.10 −1.54 0.693 0.75 0.93 4609 NM_002467 MYC −2.13 −1.70 0.7400.67 1.11 4624 NM_002471 MYH6 −1.77 −0.79 0.731 0.89 0.82 4635 NM_002476MYL4 −2.43 −1.77 0.708 0.71 1.00 85366 NM_033118 MYLK2 −1.59 −0.91 0.6980.86 0.81 23026 XM_028522 MYO16 −1.91 −1.26 0.700 0.80 0.88 4666NM_005594 NACA −1.55 −1.48 0.740 0.78 0.95 23148 XM_166571 NACAD −2.13−1.70 0.662 0.73 0.91 54187 NM_018946 NANS −1.77 −1.28 0.694 0.82 0.8423397 NM_015341 NCAPH −1.94 −1.74 0.690 0.72 0.96 23154 NM_014284 NCDN−2.18 −1.76 0.655 0.72 0.91 56926 NM_020170 NCLN −4.19 −3.59 0.320 0.440.72 4536 NM_173709 ND2 −1.53 −1.67 0.694 0.74 0.93 54820 NM_017668 NDE1−3.86 −3.41 0.260 0.44 0.60 81565 NM_030808 NDEL1 −1.84 −1.52 0.649 0.750.87 27158 NM_014434 NDOR1 −1.79 −0.76 0.680 0.88 0.77 10397 NM_006096NDRG1 −1.87 −1.21 0.641 0.80 0.80 55967 NM_018838 NDUFA12 −1.51 −1.770.669 0.73 0.91 4700 NM_002490 NDUFA6 −1.84 −0.86 0.691 0.87 0.79 4707NM_004545 NDUFB1 −2.24 −1.69 0.623 0.75 0.83 4734 NM_006154 NEDD4 −1.85−1.64 0.726 0.73 0.99 79858 NM_024800 NEK11 −1.58 −0.52 0.695 0.92 0.75284086 NM_178170 NEK8 −2.10 −1.77 0.587 0.73 0.80 349565 NM_178177NMNAT3 −1.93 −1.14 0.687 0.83 0.82 283820 NM_173614 NOMO2 −1.62 −1.170.664 0.76 0.87 9520 NM_006310 NPEPPS −2.82 −2.20 0.613 0.65 0.94 23467NM_014293 NPTXR −1.90 −1.63 0.732 0.73 1.00 2494 NM_003822 NR5A2 −2.24−2.51 0.720 0.61 1.18 80023 NM_024958 NRSN2 −1.60 −0.46 0.716 0.92 0.7825936 NM_015471 NSL1 −1.66 −0.13 0.739 0.98 0.76 4923 NM_002531 NTSR1−1.93 −2.23 0.633 0.63 1.00 170685 NM_153183 NUDT10 −1.73 −1.57 0.7000.75 0.93 11051 NM_007006 NUDT21 −2.43 −1.19 0.652 0.80 0.81 23279XM_113678 NUP160 −2.11 −1.27 0.666 0.80 0.84 4928 NM_005387 NUP98 −2.55−2.58 0.694 0.58 1.20 55916 NM_018698 NXT2 −2.18 −2.94 0.517 0.57 0.9111054 NM_007346 OGFR −2.00 −1.99 0.749 0.69 1.08 611 NM_001708 OPN1SW−2.05 −2.27 0.605 0.63 0.96 10133 NM_021980 OPTN −1.65 −2.10 0.683 0.651.05 4991 NM_002548 OR1D2 −1.53 −1.59 0.706 0.74 0.96 8387 NM_003553OR1E1 −1.97 −2.21 0.621 0.64 0.97 26211 NM_012369 OR2F1 −1.86 −2.110.653 0.66 0.99 114881 NM_017731 OSBPL7 −1.55 −1.46 0.688 0.78 0.8864172 NM_022353 OSGEPL1 −1.75 −0.39 0.709 0.94 0.75 150677 NM_148961OTOS −2.80 −1.96 0.567 0.68 0.83 27199 NM_080818 OXGR1 −1.87 −1.27 0.6570.80 0.82 27334 NM_014499 P2RY10 −1.61 −1.34 0.695 0.78 0.89 8106NM_004643 PABPN1 −1.54 −1.07 0.705 0.82 0.86 85315 NM_133367 PAQR8 −2.37−1.44 0.612 0.74 0.83 344838 NM_198504 PAQR9 −2.02 −3.47 0.667 0.49 1.355092 NM_000281 PCBD1 −2.10 −1.88 0.596 0.69 0.87 57060 NM_020418 PCBP4−2.01 −1.02 0.700 0.86 0.82 5116 NM_006031 PCNT −2.14 −2.64 0.639 0.611.05 9141 NM_004708 PDCD5 −3.14 −3.53 0.398 0.42 0.96 5145 NM_000440PDE6A −1.80 −1.28 0.655 0.79 0.83 8622 NM_003719 PDE8B −2.35 −0.89 0.6930.87 0.80 5159 NM_002609 PDGFRB −1.80 −1.58 0.642 0.74 0.87 204474NM_174924 PDILT −2.98 −1.71 0.530 0.70 0.75 10630 NM_006474 PDPN −1.50−1.56 0.640 0.74 0.86 23037 NM_015022 PDZD2 −1.83 −0.91 0.720 0.86 0.8410455 NM_006117 PECI −1.63 −1.10 0.688 0.82 0.84 27043 NM_014389 PELP1−2.08 −1.98 0.602 0.67 0.90 64065 NM_022121 PERP −1.73 −2.24 0.668 0.631.06 92960 NM_080662 PEX11G −2.18 −4.07 0.585 0.41 1.42 5207 NM_002625PFKFB1 −2.30 −2.34 0.546 0.62 0.88 192111 NM_138575 PGAM5 −1.59 −1.000.720 0.83 0.86 80055 NM_024989 PGAP1 −1.74 −1.11 0.684 0.81 0.85 5232NM_138733 PGK2 −2.53 −2.32 0.501 0.63 0.80 221692 XM_166420 PHACTR1−1.86 −1.34 0.704 0.76 0.92 112885 NM_138415 PHF21B −1.51 −2.39 0.6970.64 1.09 84844 NM_032758 PHF5A −2.27 −1.75 0.627 0.67 0.93 51588NM_015897 PIAS4 −2.04 −1.34 0.748 0.74 1.00 5291 NM_006219 PIK3CB −1.87−1.64 0.640 0.73 0.88 26034 NM_015553 PIP3-E −1.57 −0.68 0.751 0.88 0.8555124 NM_018068 PIWIL2 −1.75 −1.21 0.665 0.80 0.83 168507 NM_138295PKD1L1 −2.07 −2.06 0.644 0.68 0.95 9033 NM_016112 PKD2L1 −1.85 −1.870.657 0.67 0.98 5570 NM_032471 PKIB −2.34 −1.95 0.538 0.69 0.78 123745NM_198442 PLA2G4E −1.99 −2.92 0.642 0.59 1.09 22874 NM_014935 PLEKHA6−2.09 −1.04 0.697 0.83 0.84 58473 NM_021200 PLEKHB1 −1.54 −0.79 0.7410.86 0.86 10979 NM_006832 PLEKHC1 −1.69 −1.32 0.676 0.78 0.86 57480XM_027307 PLEKHG1 −1.60 −0.94 0.742 0.85 0.87 5356 NM_002669 PLRG1 −2.11−1.42 0.738 0.78 0.95 5359 NM_021105 PLSCR1 −1.81 −1.73 0.665 0.69 0.9657048 NM_020360 PLSCR3 −1.83 −1.14 0.660 0.80 0.82 5373 NM_000303 PMM2−3.21 −2.03 0.594 0.60 1.00 5395 NM_000535 PMS2 −1.83 −1.79 0.735 0.740.99 25953 NM_015488 PNKD −1.72 −1.63 0.669 0.73 0.92 11284 NM_007254PNKP −1.56 −1.68 0.688 0.73 0.95 5430 NM_000937 POLR2A −5.91 −5.55 0.1880.13 1.46 5432 NM_002694 POLR2C −2.85 −2.66 0.523 0.61 0.86 5433NM_004805 POLR2D −5.28 −2.62 0.332 0.48 0.69 5438 NM_006233 POLR2I −2.04−1.29 0.656 0.81 0.81 5439 NM_006234 POLR2J −2.71 −3.22 0.544 0.52 1.04246721 NM_032958 POLR2J2 −2.01 −2.41 0.684 0.60 1.14 5441 NM_021128POLR2L −1.95 −1.19 0.672 0.83 0.81 171568 NM_138338 POLR3H −2.16 −2.140.624 0.66 0.95 10940 NM_015029 POP1 −4.24 −3.02 0.390 0.50 0.78 5456NM_000307 POU3F4 −2.32 −1.82 0.713 0.65 1.10 5498 NM_000309 PPOX −2.04−1.50 0.741 0.70 1.06 23645 NM_014330 PPP1R15A −2.20 −1.48 0.698 0.770.91 5534 NM_000945 PPP3R1 −2.89 −1.67 0.603 0.74 0.82 343070 XM_291638PRAMEF9 −2.37 −1.86 0.613 0.73 0.84 59336 NM_021620 PRDM13 −2.48 −2.630.524 0.57 0.93 56978 NM_020226 PRDM8 −1.63 −1.79 0.687 0.70 0.98 5550NM_002726 PREP −1.97 −1.68 0.668 0.75 0.89 5553 NM_002728 PRG2 −2.27−2.72 0.617 0.60 1.03 166336 NM_198859 PRICKLE2 −2.13 −2.68 0.632 0.581.09 5564 NM_006253 PRKAB1 −2.19 −2.65 0.580 0.56 1.03 5565 NM_005399PRKAB2 −1.80 −1.41 0.656 0.77 0.86 5567 NM_002731 PRKACB −1.80 −0.950.639 0.84 0.76 53632 NM_017431 PRKAG3 −1.52 −1.29 0.702 0.79 0.89 5617NM_000948 PRL −2.06 −1.25 0.741 0.80 0.92 23627 NM_012409 PRND −2.02−2.26 0.612 0.63 0.98 5625 NM_016335 PRODH −2.35 −2.40 0.702 0.52 1.3458510 NM_021232 PRODH2 −3.37 −1.97 0.574 0.61 0.94 8559 NM_003675 PRPF18−2.48 −1.27 0.652 0.82 0.80 27339 NM_014502 PRPF19 −1.79 −2.09 0.6820.68 1.01 26121 NM_015629 PRPF31 −1.88 −1.33 0.639 0.78 0.82 5698NM_002800 PSMB9 −2.94 −1.80 0.627 0.64 0.98 5714 NM_002812 PSMD8 −4.26−4.07 0.182 0.33 0.55 5740 NM_000961 PTGIS −2.23 −0.75 0.717 0.85 0.845757 NM_002823 PTMA −2.62 −2.15 0.571 0.63 0.91 5805 NM_000317 PTS −1.50−0.92 0.738 0.84 0.88 9543 NM_004884 PUNC −2.25 −0.91 0.710 0.86 0.8284074 NM_032134 QRICH2 −1.67 −2.79 0.686 0.55 1.26 5768 NM_002826 QSCN6−2.59 −2.10 0.570 0.63 0.90 26056 NM_015470 RAB11FIP5 −1.64 −1.48 0.7410.76 0.97 9609 NM_004914 RAB36 −1.95 −1.94 0.747 0.71 1.05 338382NM_177403 RAB7B −1.84 −1.17 0.699 0.83 0.84 4218 NM_005370 RAB8A −1.62−1.88 0.689 0.69 1.00 5876 NM_004582 RABGGTB −2.34 −1.78 0.704 0.65 1.095885 NM_006265 RAD21 −1.83 −1.09 0.744 0.85 0.88 5888 NM_002875 RAD51−1.87 −2.50 0.722 0.59 1.22 10411 NM_006105 RAPGEF3 −2.07 −1.79 0.6960.76 0.92 9462 NM_004841 RASAL2 −2.61 −2.66 0.661 0.60 1.09 10235NM_005825 RASGRP2 −4.38 −2.56 0.419 0.50 0.84 8045 NM_003475 RASSF7−1.74 −2.48 0.722 0.62 1.16 10741 NM_006606 RBBP9 −4.88 −3.74 0.190 0.350.54 79171 NM_024321 RBM42 −1.73 −1.83 0.711 0.71 1.00 27303 NM_014483RBMS3 −1.55 −1.60 0.733 0.72 1.02 5950 NM_006744 RBP4 −2.25 −1.67 0.6270.71 0.88 9978 NM_014248 RBX1 −1.94 −1.42 0.715 0.77 0.93 9986 NM_005133RCE1 −2.36 −1.98 0.700 0.61 1.16 57333 NM_020650 RCN3 −2.49 −3.01 0.5960.53 1.12 25807 NM_012265 RHBDD3 −1.74 −1.10 0.726 0.82 0.89 57127NM_020407 RHBG −1.83 −1.13 0.699 0.83 0.85 391 NM_001665 RHOG −1.61−0.56 0.738 0.91 0.81 171177 NM_133639 RHOV −1.52 −1.86 0.716 0.67 1.0783732 NM_031480 RIOK1 −2.26 −1.70 0.575 0.76 0.76 84659 NM_032572 RNASE7−1.87 −1.61 0.691 0.71 0.97 140432 NM_178861 RNF113B −1.76 −1.42 0.6430.78 0.83 79845 NM_024787 RNF122 −2.03 −1.15 0.652 0.82 0.79 158763NM_144967 RP13-102H20.1 −2.11 −3.99 0.673 0.37 1.84 6134 NM_006013 RPL10−1.58 −2.08 0.736 0.64 1.15 4736 NM_007104 RPL10A −1.51 −2.41 0.745 0.651.15 11224 NM_007209 RPL35 −1.83 −1.69 0.701 0.73 0.96 6125 NM_000969RPL5 −1.62 −2.14 0.734 0.63 1.17 6217 NM_001020 RPS16 −1.63 −1.69 0.7310.71 1.03 6187 NM_002952 RPS2 −2.72 −1.81 0.676 0.70 0.96 6227 NM_001024RPS21 −3.01 −1.89 0.641 0.69 0.93 6188 NM_001005 RPS3 −2.44 −1.62 0.7100.73 0.97 6193 NM_001009 RPS5 −2.80 −2.12 0.666 0.65 1.02 6196 NM_021135RPS6KA2 −1.59 −1.25 0.720 0.82 0.87 3921 NM_002295 RPSA −2.47 −2.350.654 0.62 1.06 64121 NM_022157 RRAGC −1.79 −1.16 0.701 0.80 0.87 6240NM_001033 RRM1 −5.53 −3.82 0.299 0.24 1.24 6241 NM_001034 RRM2 −6.12−4.07 0.226 0.19 1.18 54700 NM_018427 RRN3 −2.99 −1.54 0.705 0.72 0.9823212 NM_015169 RRS1 −1.77 −2.08 0.720 0.67 1.08 349667 NM_178570RTN4RL2 −2.87 −1.66 0.596 0.73 0.82 860 NM_004348 RUNX2 −2.50 −1.010.694 0.81 0.86 113174 NM_138421 SAAL1 −1.86 −1.48 0.623 0.78 0.80 6299NM_002968 SALL1 −2.70 −2.24 0.626 0.68 0.93 9092 NM_005146 SART1 −1.70−0.64 0.708 0.89 0.80 6307 NM_006745 SC4MOL −2.57 −1.79 0.674 0.64 1.05113178 NM_079834 SCAMP4 −2.18 −1.71 0.559 0.74 0.75 92344 NM_152281SCYL1BP1 −1.65 −1.53 0.688 0.78 0.88 6392 NM_003002 SDHD −2.06 −1.210.642 0.79 0.81 284904 NM_174977 SEC14L4 −1.54 −1.82 0.719 0.74 0.9781929 NM_031216 SEH1L −1.77 −1.12 0.673 0.80 0.84 22929 NM_012247 SEPHS1−1.51 −0.68 0.731 0.90 0.81 871 NM_001235 SERPINH1 −2.63 −0.82 0.6510.84 0.78 5274 NM_005025 SERPINI1 −2.31 −2.56 0.702 0.62 1.13 54093NM_017438 SETD4 −2.82 −2.84 0.517 0.61 0.85 387893 NM_020382 SETD8 −1.57−1.54 0.708 0.73 0.97 387893 NM_020382 SETD8 −2.15 −1.49 0.728 0.70 1.03124925 NM_178860 SEZ6 −2.75 −2.58 0.507 0.63 0.80 10291 NM_005877 SF3A1−2.45 −2.31 0.657 0.67 0.99 10946 NM_006802 SF3A3 −3.21 −2.27 0.551 0.670.82 23451 NM_012433 SF3B1 −2.80 −3.80 0.561 0.39 1.43 10992 XM_290506SF3B2 −2.97 −3.05 0.572 0.50 1.15 23450 NM_012426 SF3B3 −2.68 −3.020.580 0.52 1.13 83443 NM_031287 SF3B5 −2.85 −2.42 0.481 0.56 0.85 6421NM_005066 SFPQ −3.20 −2.15 0.554 0.69 0.80 6428 NM_003017 SFRS3 −2.44−1.02 0.657 0.85 0.77 6433 NM_152235 SFRS8 −2.02 −0.27 0.716 0.95 0.75118980 NM_178858 SFXN2 −2.36 −2.38 0.577 0.67 0.86 151648 NM_138484SGOL1 −2.79 −3.45 0.567 0.46 1.24 10044 NM_005489 SH2D3C −3.26 −3.200.579 0.52 1.10 79628 NM_024577 SH3TC2 −1.87 −1.78 0.673 0.73 0.93 25942NM_015477 SIN3A −2.22 −1.74 0.679 0.75 0.91 140885 NM_080792 SIRPA −1.58−0.36 0.737 0.94 0.79 284759 XM_209363 SIRPB2 −2.09 −2.32 0.620 0.670.93 51804 NM_017420 SIX4 −2.08 −1.56 0.746 0.70 1.07 147912 NM_175875SIX5 −1.60 −1.10 0.716 0.84 0.85 4990 NM_007374 SIX6 −2.27 −1.42 0.7220.72 1.00 7884 NM_006527 SLBP −3.33 −2.00 0.536 0.71 0.75 10723NM_006598 SLC12A7 −3.67 −2.99 0.473 0.51 0.93 117247 NM_018593 SLC16A10−1.99 −3.01 0.642 0.58 1.11 123041 NM_153646 SLC24A4 −2.17 −2.11 0.6150.71 0.87 10166 NM_014252 SLC25A15 −1.52 −2.07 0.719 0.63 1.14 115286NM_173471 SLC25A26 −2.52 −2.99 0.522 0.57 0.91 64924 NM_022902 SLC30A5−1.56 −1.73 0.739 0.72 1.02 10463 NM_006345 SLC30A9 −1.93 −2.12 0.5360.65 0.82 285641 NM_181774 SLC36A3 −1.92 −1.43 0.653 0.79 0.82 29986NM_014579 SLC39A2 −1.68 −1.73 0.710 0.73 0.97 283375 NM_173596 SLC39A5−2.26 −1.71 0.506 0.65 0.78 23446 NM_022109 SLC44A1 −2.56 −2.65 0.5990.58 1.04 85414 NM_033102 SLC45A3 −1.99 −2.12 0.674 0.61 1.10 11309NM_007256 SLCO2B1 −1.99 −1.67 0.714 0.73 0.98 6603 NM_003077 SMARCD2−2.09 −0.28 0.740 0.94 0.79 23049 NM_014006 SMG1 −1.75 −2.13 0.685 0.700.98 6606 NM_000344 SMN1 −2.54 −1.74 0.662 0.66 1.01 6607 NM_017411 SMN2−1.65 −1.57 0.732 0.73 1.01 55234 NM_018225 SMU1 −3.35 −3.96 0.208 0.400.52 80725 NM_025248 SNIP −1.64 −1.09 0.699 0.81 0.87 51429 NM_016224SNX9 −2.56 −1.33 0.646 0.78 0.83 8435 NM_003578 SOAT2 −2.47 −2.23 0.6880.56 1.23 6651 NM_003103 SON −5.33 −3.44 0.253 0.51 0.50 10615 NM_006461SPAG5 −1.81 −1.73 0.572 0.72 0.80 83893 NM_031955 SPATA16 −1.96 −1.470.627 0.76 0.82 219938 NM_174927 SPATA19 −1.85 −1.75 0.708 0.70 1.0184651 NM_032566 SPINK7 −1.65 −1.58 0.729 0.71 1.02 81848 NM_030964 SPRY4−1.68 −1.63 0.690 0.70 0.99 136853 NM_080744 SRCRB4D −2.36 −2.26 0.6950.66 1.05 253017 XM_171068 SRD5A2L2 −2.28 −1.31 0.639 0.78 0.82 6756NM_005635 SSX1 −2.26 −0.88 0.723 0.83 0.87 57559 NM_020799 STAMBPL1−1.78 −0.89 0.670 0.84 0.80 134429 NM_139164 STARD4 −2.01 −1.44 0.5970.78 0.77 80765 NM_030574 STARD5 −1.69 −1.50 0.686 0.74 0.93 10273NM_005861 STUB1 −2.00 −0.64 0.699 0.90 0.78 6804 NM_004603 STX1A −2.18−0.72 0.732 0.88 0.83 56670 NM_033050 SUCNR1 −1.51 −1.94 0.724 0.70 1.036829 NM_003169 SUPT5H −2.87 −2.40 0.527 0.58 0.90 6830 NM_003170 SUPT6H−2.94 −2.08 0.619 0.69 0.90 9412 NM_004264 SURB7 −2.22 −2.07 0.714 0.691.03 136306 NM_174959 SVOPL −2.43 −2.66 0.522 0.58 0.89 25949 NM_015484SYF2 −2.96 −3.00 0.535 0.52 1.04 54843 NM_032379 SYTL2 −2.23 −1.98 0.6340.73 0.87 10460 NM_006342 TACC3 −2.06 −0.78 0.663 0.86 0.77 11138NM_007063 TBC1D8 −2.10 −1.87 0.698 0.69 1.01 90665 NM_033284 TBL1Y −2.12−2.38 0.571 0.65 0.87 6911 NM_004608 TBX6 −2.44 −1.22 0.697 0.77 0.918557 NM_003673 TCAP −3.41 −1.87 0.591 0.69 0.85 200132 NM_152665TCTEX1D1 −1.88 −1.83 0.673 0.72 0.94 7011 NM_007110 TEP1 −1.83 −1.750.749 0.72 1.03 10227 NM_001120 TETRAN −1.55 −1.25 0.626 0.80 0.78 56159NM_031276 TEX11 −1.72 −1.31 0.721 0.80 0.91 7018 NM_001063 TP −1.90−2.37 0.730 0.66 1.11 7069 NM_003251 THRSP −1.60 −0.53 0.744 0.92 0.8110333 NM_006068 TLR6 −1.68 −1.28 0.653 0.82 0.80 117532 NM_080751 TMC2−1.55 −1.34 0.714 0.81 0.88 23671 NM_016192 TMEFF2 −1.70 −1.50 0.7330.76 0.97 200728 NM_198276 TMEM17 −2.02 −1.07 0.646 0.82 0.79 80775NM_030577 TMEM177 −2.49 −1.63 0.542 0.72 0.75 199964 NM_182532 TMEM61−1.97 −1.35 0.660 0.78 0.84 137835 NM_144649 TMEM71 −1.80 −1.16 0.6500.83 0.78 388730 NM_203376 TMEM81 −1.91 −1.25 0.565 0.74 0.76 388364NM_206832 TMIGD1 −2.12 −2.28 0.610 0.60 1.01 28983 NM_014058 TMPRSS11E−1.87 −0.86 0.659 0.84 0.78 23495 NM_012452 TNFRSF13B −2.22 −1.33 0.6880.78 0.88 8711 NM_003985 TNK1 −1.64 −1.81 0.682 0.74 0.92 85456NM_033396 TNKS1BP1 −2.17 −1.33 0.643 0.75 0.86 7134 NM_003280 TNNC1−2.35 −1.45 0.688 0.72 0.96 30000 NM_013433 TNPO2 −3.20 −3.50 0.489 0.461.06 26058 NM_015575 TNRC15 −1.63 −1.57 0.744 0.76 0.98 57690 NM_018996TNRC6C −2.03 −1.25 0.656 0.78 0.84 7153 NM_001067 TOP2A −2.28 −3.060.635 0.53 1.20 7163 NM_005079 TPD52 −2.30 −1.61 0.629 0.75 0.84 93492NM_130785 TPTE2 −1.68 −1.09 0.741 0.82 0.90 22974 NM_012112 TPX2 −5.55−3.99 0.202 0.34 0.59 11139 NM_007064 TRAD −1.84 −1.79 0.632 0.74 0.857185 NM_005658 TRAF1 −1.80 −1.64 0.713 0.75 0.95 84231 NM_032271 TRAF7−1.97 −1.44 0.624 0.76 0.82 58485 NM_021210 TRAPPC1 −1.60 −1.48 0.7340.75 0.98 54209 NM_018965 TREM2 −2.49 −3.33 0.653 0.46 1.43 80128NM_025058 TRIM46 −1.58 −1.15 0.709 0.79 0.89 84676 NM_032588 TRIM63−1.87 −1.65 0.693 0.70 0.99 81786 NM_033342 TRIM7 −1.64 −1.82 0.696 0.671.04 54802 NM_017646 TRIT1 −1.75 −0.52 0.714 0.93 0.77 54822 NM_017672TRPM7 −1.63 −0.38 0.711 0.94 0.75 29122 NM_013270 TSP50 −1.94 −1.420.658 0.78 0.84 26262 NM_130465 TSPAN17 −2.78 −2.50 0.558 0.62 0.9084630 XM_166453 TTBK1 −1.86 −1.66 0.669 0.76 0.87 54902 NM_017775 TTC19−1.85 −1.20 0.746 0.81 0.92 83538 NM_031421 TTC25 −1.65 −1.82 0.690 0.700.98 7272 NM_003318 TTK −1.78 −2.34 0.670 0.67 1.00 26140 NM_015644TTLL3 −2.13 −2.96 0.663 0.52 1.27 57348 NM_020659 TTYH1 −1.58 −1.890.742 0.71 1.05 10376 NM_006082 TUBA1B −2.55 −1.42 0.650 0.78 0.84 84790NM_032704 TUBA1C −2.60 −1.83 0.575 0.67 0.86 7283 NM_001070 TUBG1 −2.08−0.55 0.714 0.91 0.78 11338 NM_007279 U2AF2 −2.96 −1.88 0.576 0.69 0.837311 NM_003333 UBA52 −2.72 −2.01 0.675 0.67 1.01 51271 NM_016525 UBAP1−1.66 −0.46 0.733 0.93 0.79 9040 NM_003969 UBE2M −1.80 −1.42 0.709 0.780.91 54658 NM_000463 UGT1A1 −1.63 −1.77 0.739 0.72 1.02 54579 NM_019078UGT1A5 −1.59 −1.19 0.716 0.84 0.86 51720 NM_016290 UIMC1 −1.98 −1.190.725 0.80 0.90 90249 XM_030300 UNC5A −2.03 −2.14 0.590 0.68 0.86 23353NM_025154 UNC84A −2.07 −1.88 0.676 0.70 0.97 85451 XM_036115 UNK −1.55−0.56 0.745 0.89 0.83 388325 NM_207103 UNQ5783 −1.95 −1.26 0.636 0.780.82 139596 NM_145052 UPRT −1.56 −1.92 0.696 0.71 0.98 27089 NM_014402UQCRQ −2.12 −2.02 0.624 0.69 0.91 9097 NM_005151 USP14 −1.83 −0.67 0.7190.89 0.80 373856 XM_036729 USP41 −1.58 −2.22 0.710 0.60 1.18 9712NM_014688 USP6NL −1.99 −3.76 0.557 0.31 1.79 10208 NM_005800 USPL1 −2.20−1.42 0.672 0.77 0.88 9686 NM_014667 VGLL4 −1.69 −2.30 0.617 0.57 1.087431 NM_003380 VIM −2.39 −1.95 0.713 0.68 1.05 8876 NM_004666 VNN1 −1.67−0.42 0.732 0.94 0.78 155382 XM_376631 VPS37D −1.84 −0.95 0.717 0.840.85 11193 NM_007187 WBP4 −1.91 −0.83 0.724 0.86 0.84 23001 NM_014991WDFY3 −2.12 −2.04 0.660 0.67 0.98 11169 NM_007086 WDHD1 −2.17 −2.190.691 0.64 1.08 91833 NM_144574 WDR20 −1.59 −1.08 0.696 0.85 0.82 79819NM_024763 WDR78 −2.50 −2.83 0.564 0.56 1.00 7465 NM_003390 WEE1 −3.71−5.41 0.316 0.23 1.37 164237 NM_172005 WFDC13 −1.53 −0.56 0.735 0.910.81 140686 NM_181522 WFDC3 −1.56 −1.00 0.684 0.83 0.82 90199 NM_130896WFDC8 −2.52 −2.56 0.494 0.62 0.79 147179 NM_133264 WIPF2 −2.23 −2.430.604 0.64 0.94 7478 NM_031933 WNT8A −2.09 −2.53 0.708 0.59 1.20 56949NM_020196 XAB2 −4.01 −3.62 0.349 0.44 0.80 170626 NM_130776 XAGE3 −2.48−2.76 0.570 0.56 1.01 7512 NM_003399 XPNPEP2 −2.25 −1.68 0.692 0.74 0.942547 NM_001469 XRCC6 −2.21 −1.88 0.697 0.71 0.99 26137 NM_015642 ZBTB20−2.12 −1.74 0.665 0.72 0.92 360023 NM_194314 ZBTB41 −1.55 −1.79 0.7190.71 1.02 84186 NM_032226 ZCCHC7 −1.91 −1.27 0.640 0.79 0.81 55146NM_018106 ZDHHC4 −1.61 −1.49 0.630 0.77 0.82 60685 NM_021943 ZFAND3−1.81 −0.95 0.695 0.84 0.83 153527 NM_144723 ZMAT2 −1.79 −0.40 0.7240.94 0.77 118490 NM_178451 ZMYND17 −1.55 −0.50 0.717 0.93 0.77 10778NM_006629 ZNF271 −1.79 −1.50 0.744 0.75 0.99 92822 NM_152287 ZNF276−2.13 −2.77 0.598 0.60 1.00 91975 NM_052860 ZNF300 −1.81 −2.22 0.6500.68 0.95 57343 NM_020657 ZNF304 −2.46 −2.14 0.600 0.67 0.90 162967XM_371190 ZNF320 −1.81 −1.18 0.684 0.81 0.85 79893 NM_024835 ZNF403−1.66 −2.55 0.713 0.61 1.18 126070 NM_152357 ZNF440 −1.64 −2.02 0.7040.72 0.98 168544 XM_095168 ZNF467 −1.61 −2.10 0.722 0.67 1.08 197407NM_152652 ZNF553 −1.75 −1.73 0.693 0.72 0.96 147837 NM_145276 ZNF563−1.55 −0.93 0.724 0.86 0.84 201514 NM_173548 ZNF584 −2.15 −1.61 0.6590.74 0.90 169270 NM_173539 ZNF596 −1.51 −0.60 0.737 0.89 0.82 80095NM_025027 ZNF606 −1.85 −1.12 0.656 0.80 0.82 23352 NM_020765 ZUBR1 −1.93−0.63 0.692 0.90 0.77

TABLE 2A Highly Selective siRNAs (SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29,33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151) Entrez Cher- GeneGene MAD MAD Fold ry ID Accession # Symbol Catalog # BPLER HMLER BPLERFold_HMLER BPLER_RSD HMLER_RSD Ratio Score 5692 NM_002796 PSMB4M-011362-00 −6.73 −2.41 0.147 0.52 0.07 0.13 0.28 4 5682 NM_002786 PSMA1M-010123-01 −5.53 −2.21 0.240 0.65 0.06 0.07 0.37 4 29127 NM_013277RACGAP1 M-008650-00 −3.61 −1.46 0.361 0.77 0.14 0.08 0.47 4 5702NM_002804 PSMC3 M-008738-01 −1.77 −1.02 0.399 0.81 0.10 0.07 0.49 4 1659NM_004941 DHX8 M-010506-01 −4.27 −1.24 0.413 0.81 0.05 0.05 0.51 4 5684NM_002788 PSMA3 M-011758-00 −1.72 −0.54 0.434 0.90 0.17 0.09 0.48 411269 NM_007242 DDX19B M-013471-00 −4.84 −2.09 0.304 0.66 0.07 0.06 0.463 22938 NM_012245 SNW1 M-012446-00 −5.45 −1.31 0.335 0.75 0.10 0.08 0.453 79441 NM_024511 C4orf15 M-018131-00 −2.52 0.55 0.560 1.09 0.09 0.030.52 3 5683 NM_002787 PSMA2 M-011757-00 −4.14 −2.79 0.205 0.54 0.15 0.010.38 2 10594 NM_006445 PRPF8 M-012252-01 −5.34 −2.64 0.246 0.62 0.220.04 0.40 2 5713 NM_002811 PSMD7 M-009621-01 −3.88 −2.86 0.256 0.53 0.080.02 0.49 2 5901 NM_006325 RAN M-010353-00 −5.67 −2.89 0.266 0.57 0.060.10 0.47 2 59286 NM_024292 UBL5 M-014320-00 −4.14 −2.33 0.337 0.64 0.110.01 0.53 2 5515 NM_002715 PPP2CA M-003598-00 −2.77 −0.87 0.457 0.860.07 0.09 0.53 2 5700 NM_002802 PSMC1 M-009578-00 −1.50 −0.45 0.476 0.910.19 0.04 0.52 2 57474 NM_020714 ZNF490 M-013937-00 −4.40 −2.66 0.2850.59 0.08 0.03 0.49 1 147015 NM_144683 DHRS13 M-008777-00 −3.66 −1.690.350 0.75 0.04 0.07 0.47 1 64763 NM_022752 ZNF574 M-007054-00 −3.52−1.41 0.425 0.79 0.15 0.11 0.54 1 91869 NM_052859 RFT1 M-018174-00 −2.820.04 0.468 1.01 0.12 0.05 0.47 1 65243 NM_023070 ZNF643 M-007056-00−3.05 −0.52 0.493 0.92 0.14 0.03 0.54 1 84922 NM_032836 FIZ1 M-015014-00−2.97 −0.32 0.514 0.94 0.07 0.05 0.55 1 23474 NM_014297 ETHE1M-012508-00 −2.19 1.03 0.641 1.17 0.25 0.05 0.55 1 80863 NM_030651 PRRT1M-016655-00 −3.57 −2.00 0.348 0.64 0.18 0.10 0.54 0 2850 NM_018971 GPR27M-005562-01 −3.35 −1.23 0.385 0.81 0.05 0.15 0.47 0 9503 NM_020411 XAGE1M-013187-00 −2.09 0.53 0.541 1.11 0.09 0.17 0.49 0

TABLE 2B Moderately Selective siRNAs Entrez Gene MAD MAD Gene IDAccession # Symbol Catalog # BPLER HMLER Fold_BPLER 84950 NM_032864PRPF38A M-014833-00 −3.64 −1.70 0.403 5431 NM_000938 POLR2B M-011187-00−4.09 −1.97 0.438 115106 NM_138443 CCDC5 M-015491-00 −2.18 −0.02 0.58311157 NM_007080 LSM6 M-019754-00 −2.83 −0.41 0.593 5708 NM_002808 PSMD2M-017212-01 −4.64 −2.67 0.343 10213 NM_005805 PSMD14 M-006024-00 −3.37−2.28 0.352 10403 NM_006101 NDC80 M-004106-00 −3.24 −2.06 0.377 79680NM_024627 C22orf29 M-016338-00 −3.47 −2.46 0.400 24148 NM_012469 PRPF6M-012821-00 −3.13 −1.23 0.508 57819 NM_021177 LSM2 M-017813-00 −2.90−0.95 0.512 9861 NM_014814 PSMD6 M-021249-00 −2.05 −0.79 0.516 91746NM_133370 YTHDC1 M-015332-00 −2.31 −0.26 0.567 57461 NM_020701 ISY1M-013894-00 −4.27 −2.95 0.306 55696 NM_018047 RBM22 M-021186-00 −2.37−1.53 0.477 151903 NM_144716 CCDC12 M-015455-00 −3.22 −1.34 0.502 83540NM_031423 NUF2 M-005289-01 −2.68 −0.90 0.515 26036 NM_015555 ZNF451M-013935-00 −3.07 −0.77 0.516 1213 NM_004859 CLTC M-004001-00 −2.02 0.050.570 2597 NM_002046 GAPDH M-004253-01 −4.30 −0.19 0.576 5211 NM_002626PFKL M-006822-00 −1.85 −0.10 0.630 55735 NM_018198 DNAJC11 M-021205-00−1.52 0.75 0.656 57835 NM_021196 SLC4A5 M-007585-00 −1.94 0.63 0.672388531 NM_207391 RGS9BP M-032069-00 −1.58 0.53 0.707 154007 NM_152551C6orf151 M-018855-00 −1.75 1.64 0.730 1315 NM_016451 COPB1 M-017940-00−3.13 −2.91 0.326 3190 NM_002140 HNRPK M-011692-00 −3.35 −2.71 0.35810114 NM_005734 HIPK3 M-004810-00 −3.97 −2.34 0.418 11325 NM_007372DDX42 M-012393-00 −4.00 −1.78 0.425 90407 NM_080652 TMEM41A M-015245-00−2.70 −1.83 0.457 9821 NM_014781 RB1CC1 M-021117-00 −3.21 −1.50 0.46651477 NM_016368 ISYNA1 M-009669-00 −1.95 −0.82 0.489 286826 NM_173083LIN9 M-018918-00 −2.90 −0.72 0.525 84975 NM_032889 MFSD5 M-018634-00−2.81 −0.38 0.540 79622 NM_024571 C16orf33 M-014370-00 −2.67 −0.58 0.5406457 NM_003027 SH3GL3 M-015728-00 −2.67 −0.55 0.544 5128 NM_002595 PCTK2M-004835-01 −2.29 −0.79 0.552 29945 NM_013367 ANAPC4 M-013642-00 −2.96−0.79 0.557 64236 NM_021630 PDLIM2 M-010731-00 −2.65 −0.34 0.563 27316NM_002139 RBMX M-011691-00 −2.43 −0.57 0.569 56931 NM_020175 DUS3LM-031942-00 −2.65 −0.71 0.571 51473 NM_016356 DCDC2 M-020868-00 −1.61−0.02 0.571 55957 NM_019104 LIN37 M-013311-00 −1.92 −0.19 0.571 4247NM_002408 MGAT2 M-011333-00 −2.06 −0.19 0.586 115361 NM_052941 GBP4M-018177-00 −2.09 −0.14 0.608 8291 NM_003494 DYSF M-003652-01 −2.13 0.640.614 57456 NM_020696 KIAA1143 M-013876-00 −2.27 0.86 0.631 255104NM_181719 TMCO4 M-018952-00 −1.74 −0.01 0.638 3763 NM_002240 KCNJ6M-006251-00 −1.88 0.12 0.654 8621 NM_003718 CDC2L5 M-004688-00 −2.340.95 0.656 89777 NM_080474 SERPINB12 M-008758-00 −1.66 0.63 0.668 26994NM_014372 RNF11 M-006971-00 −1.86 1.11 0.671 91978 NM_033513 C19orf20M-023892-00 −1.69 0.87 0.684 192134 NM_138706 B3GNT6 M-016388-00 −1.800.45 0.691 154075 NM_152552 SAMD3 M-015449-00 −1.99 0.68 0.692 2838NM_005290 GPR15 M-005550-00 −1.58 0.72 0.708 79443 NM_024513 FYCO1M-014350-00 −1.67 0.86 0.714 150946 XM_097977 FAM59B M-022647-00 −1.850.68 0.714 59269 NM_024503 HIVEP3 M-014345-00 −5.23 −2.35 0.306 554NM_000054 AVPR2 M-005432-00 −3.26 −2.16 0.401 57621 NM_020861 ZBTB2M-014129-00 −3.50 −1.59 0.412 57701 XM_035497 KIAA1602 M-026953-00 −3.29−1.54 0.446 148930 NM_182516 L5 M-018573-00 −2.89 −1.47 0.486 285676NM_182594 ZNF454 M-018860-00 −2.78 −0.94 0.494 1633 NM_000788 DCKM-006710-00 −3.39 −1.32 0.502 8335 NM_003513 HIST1H2AB M-017596-00 −1.78−1.23 0.504 84458 XM_050988 LCOR M-026303-00 −2.98 −0.81 0.511 11281NM_007252 POU6F2 M-019645-00 −3.27 −0.75 0.529 8653 NM_004660 DDX3YM-011904-00 −1.66 −1.17 0.537 1175 NM_004069 AP2S1 M-011833-00 −1.99−0.34 0.562 83787 NM_031905 ARMC10 M-018188-00 −2.13 0.45 0.596 9085NM_004680 CDY1 M-008916-00 −1.86 −0.49 0.599 285888 XM_376727 CNPY1M-028750-00 −2.15 0.40 0.612 117178 NM_014021 SSX2IP M-020361-00 −2.15−0.09 0.615 1769 NM_001371 DNAH8 M-010075-00 −1.78 −0.24 0.621 149954NM_182519 C20orf186 M-009125-00 −1.71 1.00 0.695 167838 XM_371849 TXLNBM-024703-00 −1.58 1.06 0.724 Entrez Cherry Gene ID Fold_HMLER BPLER_RSDHMLER_RSD Ratio Score 84950 0.69 0.05 0.02 0.58 4  5431 0.69 0.04 0.010.63 4 115106  1.00 0.21 0.02 0.59 4 11157 0.93 0.02 0.08 0.64 4  57080.61 0.16 0.04 0.56 3 10213 0.62 0.06 0.10 0.56 3 10403 0.66 0.07 0.020.57 3 79680 0.62 0.05 0.02 0.64 3 24148 0.81 0.20 0.13 0.62 3 578190.84 0.09 0.05 0.61 3  9861 0.87 0.05 0.08 0.59 3 91746 0.96 0.19 0.060.59 3 57461 0.54 0.07 0.06 0.56 2 55696 0.78 0.08 0.03 0.61 2 151903 0.78 0.14 0.15 0.64 2 83540 0.84 0.06 0.05 0.62 2 26036 0.86 0.19 0.170.60 2  1213 1.01 0.05 0.00 0.57 2  2597 0.97 0.11 0.09 0.60 2  52110.98 0.17 0.02 0.64 2 55735 1.11 0.16 0.03 0.59 2 57835 1.11 0.21 0.060.61 2 388531  1.10 0.07 0.06 0.64 2 154007  1.26 0.10 0.02 0.58 2  13150.54 0.24 0.09 0.61 1  3190 0.55 0.12 0.08 0.65 1 10114 0.68 0.07 0.020.61 1 11325 0.71 0.04 0.04 0.60 1 90407 0.73 0.08 0.04 0.63 1  98210.74 0.18 0.04 0.63 1 51477 0.87 0.18 0.07 0.57 1 286826  0.90 0.08 0.070.59 1 84975 0.93 0.10 0.05 0.58 1 79622 0.91 0.07 0.03 0.59 1  64570.90 0.15 0.03 0.60 1  5128 0.87 0.05 0.06 0.64 1 29945 0.87 0.06 0.040.64 1 64236 0.95 0.10 0.03 0.60 1 27316 0.91 0.16 0.03 0.62 1 569310.89 0.08 0.02 0.64 1 51473 1.00 0.19 0.19 0.57 1 55957 0.97 0.21 0.020.59 1  4247 0.97 0.02 0.07 0.60 1 115361  0.99 0.15 0.15 0.62 1  82911.12 0.11 0.08 0.55 1 57456 1.14 0.14 0.06 0.56 1 255104  0.99 0.13 0.040.64 1  3763 1.04 0.16 0.14 0.63 1  8621 1.13 0.03 0.08 0.58 1 897771.09 0.07 0.04 0.61 1 26994 1.17 0.01 0.07 0.57 1 91978 1.13 0.09 0.040.61 1 192134  1.07 0.17 0.07 0.65 1 154075  1.11 0.09 0.06 0.62 1  28381.11 0.09 0.03 0.64 1 79443 1.13 0.05 0.01 0.63 1 150946  1.10 0.20 0.070.65 1 59269 0.54 0.10 0.05 0.57 0  554 0.66 0.10 0.14 0.60 0 57621 0.730.09 0.02 0.56 0 57701 0.74 0.11 0.04 0.60 0 148930  0.78 0.04 0.08 0.620 285676  0.87 0.03 0.21 0.57 0  1633 0.82 0.05 0.01 0.61 0  8335 0.830.07 0.01 0.61 0 84458 0.85 0.10 0.03 0.60 0 11281 0.88 0.16 0.08 0.60 0 8653 0.83 0.08 0.05 0.64 0  1175 0.94 0.19 0.09 0.60 0 83787 1.07 0.120.06 0.56 0  9085 0.93 0.04 0.12 0.64 0 285888  1.06 0.05 0.08 0.58 0117178  0.98 0.01 0.04 0.63 0  1769 0.96 0.14 0.01 0.65 0 149954  1.150.11 0.02 0.61 0 167838  1.16 0.14 0.13 0.62 0

TABLE 2C Modestly Selective siRNAs Gene Accession # Catalog # SymbolMAD_BPLER MAD_HMLER Fold_BPLER NM_002696 M-011357-00 POLR2G −3.59 −1.680.507 NM_018097 M-021161-00 CEP27 −1.99 −1.76 0.522 XM_058073M-010646-00 NUP205 −2.88 −1.57 0.544 XM_375557 M-031204-00 C19orf29−2.24 −0.36 0.625 NM_001253 M-011237-00 CDC5L −1.60 −0.87 0.654NM_006292 M-003549-01 TSG101 −2.45 0.23 0.684 NM_016937 M-020856-00POLA1 −2.01 −0.17 0.724 NM_032916 M-015046-00 FAM86B1 −2.54 −2.05 0.491NM_001237 M-003205-02 CCNA2 −2.79 −1.39 0.499 NM_016047 M-020260-00SF3B14 −2.97 −2.06 0.500 NM_007364 M-008051-00 TMED3 −2.93 −1.28 0.537NM_014347 M-006964-00 ZNF324 −2.91 −1.05 0.543 NM_007263 M-017632-00COPE −3.15 −1.43 0.547 NM_005063 M-005061-01 SCD −3.24 −1.58 0.555NM_032425 M-022058-00 KIAA1822 −2.67 −1.27 0.563 NM_020860 M-013166-00STIM2 −2.39 −0.99 0.597 NM_018242 M-013347-00 SLC47A1 −1.62 −0.71 0.613NM_004336 M-004102-00 BUB1 −2.62 −0.62 0.617 NM_152655 M-016432-00ZNF585A −2.06 −0.70 0.642 NM_182640 M-019184-00 MRPS9 −2.00 −0.45 0.669NM_022830 M-014221-00 TUT1 −1.91 −0.07 0.680 XM_370928 M-022880-00TBC1D24 −1.97 −0.32 0.680 NM_152374 M-016893-00 FLJ38984 −1.50 −0.230.693 NM_006590 M-006087-00 USP39 −2.13 −0.04 0.695 NM_016200M-017030-00 LSM8 −1.76 −0.26 0.700 NM_003981 M-019491-00 PRC1 −3.08−2.40 0.409 NM_020882 M-024094-00 COL20A1 −3.47 −2.58 0.416 NM_021974M-004723-00 POLR2F −3.46 −2.98 0.417 NM_018271 M-020368-00 FLJ10916−2.39 −2.48 0.440 XM_378175 M-003993-01 LOC124446 −3.04 −2.49 0.458NM_014456 M-004438-02 PDCD4 −2.81 −2.25 0.462 NM_152496 M-016951-00MANEAL −3.02 −2.41 0.464 NM_174931 M-017855-00 CCDC75 −3.33 −2.06 0.474XM_371354 M-022129-00 KIF26B −1.95 −1.15 0.536 NM_004739 M-008482-00MTA2 −3.71 −1.07 0.545 NM_198080 M-021432-00 MSRB3 −2.72 −0.86 0.571NM_000792 M-011170-00 DIO1 −1.96 −1.39 0.573 NM_013366 M-003200-02ANAPC2 −2.80 −1.31 0.580 NM_001746 M-003636-02 CANX −1.92 −1.38 0.587NM_148571 M-013182-00 MRPL27 −1.57 −1.17 0.587 NM_002255 M-018983-00KIR2DL4 −2.02 −0.98 0.593 NM_024762 M-014438-00 ZNF552 −2.35 −1.30 0.595NM_052946 M-015170-00 NOSTRIN −2.06 −0.69 0.604 NM_002636 M-011353-00PHF1 −3.21 −0.72 0.606 NM_015046 M-021420-00 SETX −2.40 −0.72 0.617NM_006204 M-007653-00 PDE6C −1.98 −0.33 0.621 NM_001225 M-004404-00CASP4 −2.36 −0.92 0.623 NM_000172 M-009827-01 GNAT1 −2.03 −0.36 0.627NM_002270 M-011308-00 TNPO1 −1.99 −0.59 0.630 NM_030969 M-015288-00TMEM14B −1.99 −0.56 0.636 NM_020689 M-007466-00 SLC24A3 −2.23 −0.750.637 NM_020395 M-015624-00 INTS12 −2.22 −0.74 0.639 NM_021640M-013747-00 C12orf10 −2.11 −0.54 0.644 NM_014750 M-016846-00 DLG7 −1.93−0.56 0.646 NM_194324 M-019295-00 MGC39900 −2.13 −0.87 0.647 NM_014420M-020520-00 DKK4 −1.90 −0.72 0.657 NM_012202 M-012804-00 GNG3 −1.86−0.62 0.658 NM_001797 M-013493-00 CDH11 −1.56 −0.69 0.663 NM_002513M-006753-00 NME3 −1.71 0.12 0.668 NM_005954 M-012728-00 MT3 −1.77 −0.160.672 NM_000322 M-011102-00 PRPH2 −1.69 −0.56 0.675 NM_021960M-004501-04 MCL1 −1.74 −0.13 0.679 NM_000131 M-005871-00 F7 −2.01 −0.060.679 NM_020380 M-015673-00 CASC5 −1.86 −0.56 0.686 NM_022304M-005630-01 HRH2 −1.68 −0.39 0.693 NM_005003 M-019897-00 NDUFAB1 −1.82−0.20 0.694 NM_032024 M-014765-00 C10orf11 −1.58 −0.06 0.702 NM_134444M-015351-00 NLRP4 −1.68 0.11 0.702 XM_371074 M-030921-00 TANC2 −1.860.40 0.709 NM_080730 M-012988-00 HOM-TES- −1.84 −0.09 0.711 103NM_005713 M-012101-00 COL4A3BP −1.96 0.03 0.715 NM_152905 M-008306-00NEDD1 −1.56 0.59 0.721 NM_020528 M-013199-00 PCBP3 −1.69 0.15 0.727NM_006156 M-020081-00 NEDD8 −1.79 0.25 0.736 NM_012141 M-012417-00 INTS6−3.48 −2.65 0.369 NM_173626 M-007490-00 SLC26A11 −3.21 −2.50 0.415NM_003301 M-005747-00 TRHR −3.02 −2.31 0.423 NM_020809 M-026514-00ARHGAP20 −3.36 −2.17 0.434 NM_004277 M-007483-00 SLC25A27 −2.81 −1.800.476 NM_016082 M-013297-01 CDK5RAP1 −3.57 −2.01 0.477 NM_030962M-014684-00 SBF2 −2.84 −1.47 0.481 NM_001538 M-011295-00 HSF4 −4.01−1.43 0.512 NM_173556 M-018459-00 CCDC83 −3.06 −1.36 0.518 XM_016548M-025053-00 CDY2B −3.04 −1.73 0.521 XM_056282 M-024572-00 LRRC62 −2.35−1.74 0.528 NM_182703 M-019312-00 ANKDD1A −2.87 −1.76 0.532 NM_005831M-010637-00 CALCOCO2 −2.43 −1.58 0.533 NM_003526 M-011446-00 HIST1H2BC−1.64 −1.61 0.541 NM_005911 M-008818-00 MAT2A −2.25 −1.21 0.547NM_022567 M-014165-00 NYX −2.34 −0.97 0.550 NM_018360 M-020815-00CXorf15 −2.01 −1.59 0.555 NM_182558 M-018912-00 C12orf36 −1.99 −0.990.556 NM_016388 M-020821-00 TRAT1 −2.24 −1.24 0.557 NM_006413M-015336-00 RPP30 −1.84 −1.38 0.558 NM_024619 M-006817-00 FN3KRP −2.98−1.53 0.562 NM_001937 M-011641-00 DPT −2.02 −1.01 0.565 XM_375187M-022211-00 NUT −1.98 −0.97 0.574 NM_178168 M-008750-00 OR10A5 −2.31−1.21 0.576 NM_020320 M-013767-00 RARS2 −2.61 −1.37 0.576 NM_003685M-009490-00 KHSRP −1.93 −1.15 0.582 NM_152603 M-016451-00 ZNF567 −2.39−1.09 0.585 XM_114430 M-024410-00 LOC202051 −2.59 −0.79 0.592 NM_198494M-027243-00 ZNF642 −2.47 −0.63 0.594 NM_152465 M-016422-00 PROCA1 −2.24−1.20 0.602 NM_152647 M-016412-00 C15orf33 −2.27 −0.50 0.604 NM_138451M-015615-00 IQCD −2.04 −1.20 0.605 NM_024011 M-004026-01 CDC2L2 −2.70−0.55 0.605 NM_024647 M-018906-00 NUP43 −2.40 −1.30 0.605 NM_152515M-018844-00 CKAP2L −2.56 −1.17 0.605 NM_207401 M-032078-00 FLJ45717−1.68 −0.82 0.611 NM_144702 M-015707-00 C1orf92 −2.17 −0.90 0.615XM_371575 M-027984-00 PRPF40A −1.75 −0.96 0.623 XM_209941 M-026255-00C9orf117 −2.01 −0.84 0.635 NM_015446 M-013961-00 AHCTF1 −2.29 −0.690.635 NM_019067 M-015743-00 GNL3L −2.24 −0.43 0.636 NM_018922M-013292-00 PCDHGB1 −1.67 −0.81 0.637 NM_032866 M-019002-00 CGNL1 −2.22−0.57 0.637 NM_213604 M-023895-00 ADAMTSL5 −2.21 −0.83 0.640 NM_018129M-009715-00 PNPO −1.55 −0.72 0.641 NM_002510 M-011741-00 GPNMB −1.52−0.76 0.641 NM_004783 M-004171-03 TAOK2 −1.82 −0.32 0.648 XM_089747M-026312-00 C10orf80 −2.25 −0.73 0.653 NM_173821 M-018155-00 FLJ33590−1.90 −0.56 0.656 NM_014891 M-017675-00 PDAP1 −1.78 −0.08 0.658NM_005301 M-005567-00 GPR35 −1.85 −0.49 0.661 NM_021049 M-010800-00MAGEA5 −1.68 −0.36 0.662 NM_002218 M-017838-00 ITIH4 −1.65 −0.33 0.668NM_000188 M-006820-01 HK1 −2.21 −0.05 0.678 NM_015338 M-012856-00 ASXL1−1.86 −0.39 0.680 NM_019070 M-017975-00 DDX49 −1.92 −0.33 0.683NM_173853 M-017757-00 KRTCAP3 −1.83 −0.34 0.683 XM_373109 M-027171-00RP11- −1.93 0.07 0.686 145H9.1 NM_005544 M-003015-01 IRS1 −1.57 −0.470.695 NM_007205 M-032280-00 TREX2 −2.22 −0.39 0.697 NM_007358M-012796-00 MTF2 −2.11 −0.40 0.699 NM_173497 M-007198-00 HECTD2 −1.66−0.39 0.705 NM_003247 M-019745-00 THBS2 −2.48 −0.31 0.705 NM_173846M-017754-00 TPPP2 −1.65 0.12 0.707 XM_030729 M-026260-00 FAM22F −1.72−0.23 0.714 NM_015690 M-005039-01 STK36 −1.55 0.46 0.718 NM_002082M-004627-01 GRK6 −1.90 0.29 0.722 NM_004673 M-007804-01 ANGPTL1 −1.880.62 0.738 NM_182755 M-019384-00 ZNF438 −1.62 0.10 0.742 CherryAccession # Fold_HMLER BPLER_RSD HMLER_RSD Ratio Score NM_002696 0.740.07 0.05 0.69 3 NM_018097 0.74 0.10 0.09 0.70 3 XM_058073 0.75 0.020.05 0.73 3 XM_375557 0.94 0.12 0.01 0.67 3 NM_001253 0.87 0.05 0.120.75 3 NM_006292 1.04 0.09 0.04 0.66 3 NM_016937 0.97 0.05 0.04 0.74 3NM_032916 0.70 0.15 0.05 0.70 2 NM_001237 0.75 0.07 0.05 0.67 2NM_016047 0.72 0.09 0.03 0.70 2 NM_007364 0.80 0.16 0.04 0.68 2NM_014347 0.84 0.19 0.07 0.65 2 NM_007263 0.77 0.06 0.03 0.71 2NM_005063 0.75 0.04 0.04 0.74 2 NM_032425 0.77 0.02 0.08 0.73 2NM_020860 0.83 0.05 0.04 0.72 2 NM_018242 0.90 0.04 0.12 0.68 2NM_004336 0.91 0.08 0.03 0.67 2 NM_152655 0.89 0.04 0.05 0.72 2NM_182640 0.92 0.06 0.07 0.72 2 NM_022830 0.99 0.11 0.00 0.69 2XM_370928 0.95 0.08 0.03 0.71 2 NM_152374 0.97 0.17 0.01 0.72 2NM_006590 0.99 0.05 0.05 0.70 2 NM_016200 0.96 0.10 0.04 0.73 2NM_003981 0.60 0.18 0.19 0.68 1 NM_020882 0.56 0.04 0.04 0.74 1NM_021974 0.56 0.08 0.08 0.74 1 NM_018271 0.62 0.16 0.12 0.71 1XM_378175 0.66 0.12 0.13 0.70 1 NM_014456 0.63 0.16 0.04 0.73 1NM_152496 0.64 0.08 0.05 0.72 1 NM_174931 0.65 0.20 0.03 0.72 1XM_371354 0.82 0.13 0.07 0.65 1 NM_004739 0.79 0.16 0.12 0.69 1NM_198080 0.85 0.09 0.08 0.67 1 NM_000792 0.78 0.22 0.07 0.74 1NM_013366 0.79 0.07 0.03 0.74 1 NM_001746 0.80 0.07 0.08 0.74 1NM_148571 0.81 0.20 0.04 0.73 1 NM_002255 0.85 0.01 0.08 0.69 1NM_024762 0.80 0.03 0.05 0.75 1 NM_052946 0.90 0.13 0.03 0.67 1NM_002636 0.86 0.10 0.12 0.71 1 NM_015046 0.89 0.08 0.17 0.70 1NM_006204 0.95 0.08 0.07 0.66 1 NM_001225 0.86 0.15 0.05 0.73 1NM_000172 0.95 0.07 0.06 0.66 1 NM_002270 0.90 0.09 0.06 0.70 1NM_030969 0.90 0.11 0.02 0.71 1 NM_020689 0.88 0.08 0.03 0.72 1NM_020395 0.88 0.13 0.03 0.72 1 NM_021640 0.90 0.04 0.13 0.71 1NM_014750 0.90 0.15 0.05 0.72 1 NM_194324 0.87 0.22 0.11 0.74 1NM_014420 0.89 0.23 0.04 0.74 1 NM_012202 0.90 0.10 0.05 0.73 1NM_001797 0.90 0.05 0.11 0.73 1 NM_002513 1.02 0.17 0.05 0.65 1NM_005954 0.97 0.04 0.08 0.69 1 NM_000322 0.91 0.03 0.15 0.74 1NM_021960 0.98 0.07 0.01 0.69 1 NM_000131 0.99 0.13 0.05 0.69 1NM_020380 0.92 0.07 0.05 0.75 1 NM_022304 0.93 0.07 0.07 0.74 1NM_005003 0.97 0.01 0.04 0.72 1 NM_032024 0.97 0.12 0.22 0.72 1NM_134444 1.01 0.17 0.04 0.69 1 XM_371074 1.05 0.20 0.09 0.67 1NM_080730 0.99 0.03 0.02 0.72 1 NM_005713 1.00 0.13 0.06 0.71 1NM_152905 1.08 0.06 0.02 0.67 1 NM_020528 1.02 0.12 0.05 0.71 1NM_006156 1.04 0.12 0.08 0.71 1 NM_012141 0.53 0.16 0.09 0.70 0NM_173626 0.64 0.08 0.03 0.65 0 NM_003301 0.62 0.05 0.05 0.68 0NM_020809 0.63 0.05 0.02 0.69 0 NM_004277 0.68 0.22 0.15 0.70 0NM_016082 0.73 0.09 0.09 0.66 0 NM_030962 0.74 0.06 0.02 0.65 0NM_001538 0.72 0.02 0.06 0.71 0 NM_173556 0.77 0.02 0.08 0.68 0XM_016548 0.71 0.10 0.03 0.74 0 XM_056282 0.74 0.08 0.13 0.71 0NM_182703 0.74 0.17 0.13 0.72 0 NM_005831 0.74 0.07 0.03 0.72 0NM_003526 0.77 0.12 0.06 0.70 0 NM_005911 0.81 0.02 0.04 0.67 0NM_022567 0.84 0.12 0.02 0.66 0 NM_018360 0.77 0.14 0.11 0.72 0NM_182558 0.79 0.13 0.21 0.71 0 NM_016388 0.83 0.20 0.06 0.67 0NM_006413 0.78 0.15 0.09 0.71 0 NM_024619 0.79 0.18 0.05 0.71 0NM_001937 0.84 0.18 0.02 0.67 0 XM_375187 0.80 0.05 0.05 0.72 0NM_178168 0.81 0.03 0.08 0.71 0 NM_020320 0.79 0.11 0.02 0.73 0NM_003685 0.82 0.15 0.13 0.71 0 NM_152603 0.83 0.10 0.06 0.71 0XM_114430 0.85 0.14 0.19 0.69 0 NM_198494 0.91 0.08 0.10 0.65 0NM_152465 0.82 0.06 0.08 0.73 0 NM_152647 0.91 0.12 0.13 0.66 0NM_138451 0.83 0.14 0.10 0.73 0 NM_024011 0.93 0.03 0.02 0.65 0NM_024647 0.81 0.09 0.03 0.75 0 NM_152515 0.81 0.22 0.12 0.75 0NM_207401 0.83 0.21 0.05 0.73 0 NM_144702 0.87 0.10 0.12 0.71 0XM_371575 0.86 0.10 0.01 0.73 0 XM_209941 0.88 0.13 0.18 0.72 0NM_015446 0.90 0.11 0.06 0.71 0 NM_019067 0.94 0.15 0.02 0.68 0NM_018922 0.89 0.05 0.18 0.71 0 NM_032866 0.90 0.23 0.11 0.71 0NM_213604 0.88 0.09 0.08 0.73 0 NM_018129 0.90 0.13 0.05 0.72 0NM_002510 0.87 0.05 0.06 0.73 0 NM_004783 0.95 0.09 0.04 0.68 0XM_089747 0.89 0.09 0.12 0.74 0 NM_173821 0.92 0.12 0.13 0.72 0NM_014891 0.99 0.11 0.06 0.67 0 NM_005301 0.92 0.19 0.18 0.72 0NM_021049 0.95 0.03 0.07 0.70 0 NM_002218 0.95 0.01 0.06 0.70 0NM_000188 0.99 0.12 0.02 0.68 0 NM_015338 0.93 0.20 0.16 0.73 0NM_019070 0.96 0.11 0.01 0.71 0 NM_173853 0.93 0.09 0.19 0.73 0XM_373109 1.01 0.21 0.12 0.68 0 NM_005544 0.93 0.06 0.05 0.75 0NM_007205 0.94 0.18 0.07 0.74 0 NM_007358 0.94 0.07 0.06 0.74 0NM_173497 0.94 0.08 0.03 0.75 0 NM_003247 0.95 0.11 0.22 0.75 0NM_173846 1.01 0.10 0.11 0.70 0 XM_030729 0.97 0.03 0.05 0.74 0NM_015690 1.07 0.18 0.01 0.67 0 NM_002082 1.04 0.05 0.01 0.69 0NM_004673 1.11 0.04 0.18 0.67 0 NM_182755 1.02 0.23 0.16 0.73 0

TABLE 3 Triple Negative Breast Cancer Gene Signature (TGS) Genes orMalignancy-Associated Response Signature (MARS) Genes Gene SEQ ID EntrezMAD MAD Fold Fold Ratio Cherry Symbol NO: Gene ID Accession # BPLERHMLER BPLER HMLER BPLER/HMLER Score PSMB4 1 5692 NM_002796 −6.73 −2.410.147 0.52 0.28 4 PSMA1 2 5682 NM_002786 −5.53 −2.21 0.240 0.65 0.37 4RACGAP1 3 29127 NM_013277 −3.61 −1.46 0.361 0.77 0.47 4 PSMC3 4 5702NM_002804 −1.77 −1.02 0.399 0.81 0.49 4 PRPF38A 5 84950 NM_032864 −3.64−1.70 0.403 0.69 0.58 4 DHX8 6 1659 NM_004941 −4.27 −1.24 0.413 0.810.51 4 PSMA3 7 5684 NM_002788 −1.72 −0.54 0.434 0.90 0.48 4 POLR2B 85431 NM_000938 −4.09 −1.97 0.438 0.69 0.63 4 CCDC5 9 115106 NM_138443−2.18 −0.02 0.583 1.00 0.59 4 LSM6 10 11157 NM_007080 −2.83 −0.41 0.5930.93 0.64 4 PRPF6 11 24148 NM_012469 −3.13 −1.23 0.508 0.81 0.62 3NUP205 12 23165 XM_058073 −2.88 −1.57 0.544 0.75 0.73 3 DDX19B 13 11269NM_007242 −4.84 −2.09 0.304 0.66 0.46 3 SNW1 14 22938 NM_012245 −5.45−1.31 0.335 0.75 0.45 3 PSMD2 15 5708 NM_002808 −4.64 −2.67 0.343 0.610.56 3 PSMD14 16 10213 NM_005805 −3.37 −2.28 0.352 0.62 0.56 3 NDC80 1710403 NM_006101 −3.24 −2.06 0.377 0.66 0.57 3 C22orf29 18 79680NM_024627 −3.47 −2.46 0.400 0.62 0.64 3 POLR2G 19 5436 NM_002696 −3.59−1.68 0.507 0.74 0.69 3 LSM2 20 57819 NM_021177 −2.90 −0.95 0.512 0.840.61 3 PSMD6 21 9861 NM_014814 −2.05 −0.79 0.516 0.87 0.59 3 CEP27 2255142 NM_018097 −1.99 −1.76 0.522 0.74 0.70 3 C4orf15 23 79441 NM_024511−2.52 0.55 0.560 1.09 0.52 3 YTHDC1 24 91746 NM_133370 −2.31 −0.26 0.5670.96 0.59 3 C19orf29 25 58509 XM_375557 −2.24 −0.36 0.625 0.94 0.67 3CDC5L 26 988 NM_001253 −1.60 −0.87 0.654 0.87 0.75 3 TSG101 27 7251NM_006292 −2.45 0.23 0.684 1.04 0.66 3 POLA1 28 5422 NM_016937 −2.01−0.17 0.724 0.97 0.74 3 UBL5 29 59286 NM_024292 −4.14 −2.33 0.337 0.640.53 2 CCNA2 30 890 NM_001237 −2.79 −1.39 0.499 0.75 0.67 2 NUF2 3183540 NM_031423 −2.68 −0.90 0.515 0.84 0.62 2 COPE 32 11316 NM_007263−3.15 −1.43 0.547 0.77 0.71 2 PSMA2 33 5683 NM_002787 −4.14 −2.79 0.2050.54 0.38 2 PSMD7 34 5713 NM_002811 −3.88 −2.86 0.256 0.53 0.49 2 RAN 355901 NM_006325 −5.67 −2.89 0.266 0.57 0.47 2 ISY1 36 57461 NM_020701−4.27 −2.95 0.306 0.54 0.56 2 PPP2CA 37 5515 NM_002715 −2.77 −0.87 0.4570.86 0.53 2 PSMC1 38 5700 NM_002802 −1.50 −0.45 0.476 0.91 0.52 2 RBM2239 55696 NM_018047 −2.37 −1.53 0.477 0.78 0.61 2 FAM86B1 40 85002NR_003494 −2.54 −2.05 0.491 0.70 0.70 2 NM_032916 (SEQ ID NO: 181)SF3B14 41 51639 NM_016047 −2.97 −2.06 0.500 0.72 0.70 2 CCDC12 42 151903NM_144716 −3.22 −1.34 0.502 0.78 0.64 2 ZNF451 43 26036 NM_015555 −3.07−0.77 0.516 0.86 0.60 2 TMED3 44 23423 NM_007364 −2.93 −1.28 0.537 0.800.68 2 ZNF324 45 25799 NM_014347 −2.91 −1.05 0.543 0.84 0.65 2 SCD 466319 NM_005063 −3.24 −1.58 0.555 0.75 0.74 2 KIAA1822 47 84439 NM_032425−2.67 −1.27 0.563 0.77 0.73 2 CLTC 48 1213 NM_004859 −2.02 0.05 0.5701.01 0.57 2 GAPDH 49 2597 NM_002046 −4.30 −0.19 0.576 0.97 0.60 2 STIM250 57620 NM_020860 −2.39 −0.99 0.597 0.83 0.72 2 SLC47A1 51 55244NM_018242 −1.62 −0.71 0.613 0.90 0.68 2 BUB1 52 699 NM_004336 −2.62−0.62 0.617 0.91 0.67 2 ZNF585A 53 199704 NM_152655 −2.06 −0.70 0.6420.89 0.72 2 DNAJC11 54 55735 NM_018198 −1.52 0.75 0.656 1.11 0.59 2MRPS9 55 64965 NM_182640 −2.00 −0.45 0.669 0.92 0.72 2 SLC4A5 56 57835NM_021196 −1.94 0.63 0.672 1.11 0.61 2 TUT1 57 64852 NM_022830 −1.91−0.07 0.680 0.99 0.69 2 TBC1D24 58 57465 NM_020705 −1.97 −0.32 0.6800.95 0.71 2 XM_370928 (SEQ ID NO: 182) FLJ38984 59 127703 NM_152374−1.50 −0.23 0.693 0.97 0.72 2 USP39 60 10713 NM_006590 −2.13 −0.04 0.6950.99 0.70 2 LSM8 61 51691 NM_016200 −1.76 −0.26 0.700 0.96 0.73 2 RGS9BP62 388531 NM_207391 −1.58 0.53 0.707 1.10 0.64 2 C6orf151 63 154007NM_152551 −1.75 1.64 0.730 1.26 0.58 2 COPB1 64 1315 NM_016451 −3.13−2.91 0.326 0.54 0.61 1 ISYNA1 65 51477 NM_016368 −1.95 −0.82 0.489 0.870.57 1 FIZ1 66 84922 NM_032836 −2.97 −0.32 0.514 0.94 0.55 1 PDLIM2 6764236 NM_021630 −2.65 −0.34 0.563 0.95 0.60 1 ANAPC2 68 29882 NM_013366−2.80 −1.31 0.580 0.79 0.74 1 MGAT2 69 4247 NM_002408 −2.06 −0.19 0.5860.97 0.60 1 MRPL27 70 51264 NM_148571 −1.57 −1.17 0.587 0.81 0.73 1KIR2DL4 71 3805 NM_002255 −2.02 −0.98 0.593 0.85 0.69 1 ETHE1 72 23474NM_014297 −2.19 1.03 0.641 1.17 0.55 1 C12orf10 73 60314 NM_021640 −2.11−0.54 0.644 0.90 0.71 1 RNF11 74 26994 NM_014372 −1.86 1.11 0.671 1.170.57 1 FAM59B 75 150946 XM_097977 −1.85 0.68 0.714 1.10 0.65 1 NEDD8 764738 NM_006156 −1.79 0.25 0.736 1.04 0.71 1 ZNF490 77 57474 NM_020714−4.40 −2.66 0.285 0.59 0.49 1 DHRS13 78 147015 NM_144683 −3.66 −1.690.350 0.75 0.47 1 HNRPK 79 3190 NM_002140 −3.35 −2.71 0.358 0.55 0.65 1PRC1 80 9055 NM_003981 −3.08 −2.40 0.409 0.60 0.68 1 COL20A1 81 57642NM_020882 −3.47 −2.58 0.416 0.56 0.74 1 POLR2F 82 5435 NM_021974 −3.46−2.98 0.417 0.56 0.74 1 DDX42 83 11325 NM_007372 −4.00 −1.78 0.425 0.710.60 1 ZNF574 84 64763 NM_022752 −3.52 −1.41 0.425 0.79 0.54 1 FLJ1091685 55258 NM_018271 −2.39 −2.48 0.440 0.62 0.71 1 TMEM41A 86 90407NM_080652 −2.70 −1.83 0.457 0.73 0.63 1 LOC124446 87 124446 XM_378175−3.04 −2.49 0.458 0.66 0.70 1 PDCD4 88 27250 NM_014456 −2.81 −2.25 0.4620.63 0.73 1 MANEAL 89 149175 NM_152496 −3.02 −2.41 0.464 0.64 0.72 1RB1CC1 90 9821 NM_014781 −3.21 −1.50 0.466 0.74 0.63 1 RFT1 91 91869NM_052859 −2.82 0.04 0.468 1.01 0.47 1 CCDC75 92 253635 NM_174931 −3.33−2.06 0.474 0.65 0.72 1 ZNF643 93 65243 NM_023070 −3.05 −0.52 0.493 0.920.54 1 LIN9 94 286826 NM_173083 −2.90 −0.72 0.525 0.90 0.59 1 KIF26B 9555083 NM- −1.95 −1.15 0.536 0.82 0.65 1 018012 XM_371354 (SEQ ID NO:183) MFSD5 96 84975 NM_032889 −2.81 −0.38 0.540 0.93 0.58 1 C16orf33 9779622 NM_024571 −2.67 −0.58 0.540 0.91 0.59 1 SH3GL3 98 6457 NM_003027−2.67 −0.55 0.544 0.90 0.60 1 MTA2 99 9219 NM_004739 −3.71 −1.07 0.5450.79 0.69 1 PCTK2 100 5128 NM_002595 −2.29 −0.79 0.552 0.87 0.64 1ANAPC4 101 29945 NM_013367 −2.96 −0.79 0.557 0.87 0.64 1 RBMX 102 27316NM_002139 −2.43 −0.57 0.569 0.91 0.62 1 DUS3L 103 56931 NM_020175 −2.65−0.71 0.571 0.89 0.64 1 MSRB3 104 253827 NM_198080 −2.72 −0.86 0.5710.85 0.67 1 DCDC2 105 51473 NM_016356 −1.61 −0.02 0.571 1.00 0.57 1LIN37 106 55957 NM_019104 −1.92 −0.19 0.571 0.97 0.59 1 DIO1 107 1733NM_000792 −1.96 −1.39 0.573 0.78 0.74 1 CANX 108 821 NM_001746 −1.92−1.38 0.587 0.80 0.74 1 ZNF552 109 79818 NM_024762 −2.35 −1.30 0.5950.80 0.75 1 NOSTRIN 110 115677 NM_052946 −2.06 −0.69 0.604 0.90 0.67 1PHF1 111 5252 NM_002636 −3.21 −0.72 0.606 0.86 0.71 1 GBP4 112 115361NM_052941 −2.09 −0.14 0.608 0.99 0.62 1 DYSF 113 8291 NM_003494 −2.130.64 0.614 1.12 0.55 1 SETX 114 23064 NM_015046 −2.40 −0.72 0.617 0.890.70 1 PDE6C 115 5146 NM_006204 −1.98 −0.33 0.621 0.95 0.66 1 CASP4 116837 NM_001225 −2.36 −0.92 0.623 0.86 0.73 1 GNAT1 117 2779 NM_000172−2.03 −0.36 0.627 0.95 0.66 1 TNPO1 118 3842 NM_002270 −1.99 −0.59 0.6300.90 0.70 1 KIAA1143 119 57456 NM_020696 −2.27 0.86 0.631 1.14 0.56 1TMEM14B 120 81853 NM_030969 −1.99 −0.56 0.636 0.90 0.71 1 SLC24A3 12157419 NM_020689 −2.23 −0.75 0.637 0.88 0.72 1 TMCO4 122 255104 NM_181719−1.74 −0.01 0.638 0.99 0.64 1 INTS12 123 57117 NM_020395 −2.22 −0.740.639 0.88 0.72 1 DLG7 124 9787 NM_014750 −1.93 −0.56 0.646 0.90 0.72 1MGC39900 125 286527 NM_194324 −2.13 −0.87 0.647 0.87 0.74 1 KCNJ6 1263763 NM_002240 −1.88 0.12 0.654 1.04 0.63 1 CDC2L5 127 8621 NM_003718−2.34 0.95 0.656 1.13 0.58 1 DKK4 128 27121 NM_014420 −1.90 −0.72 0.6570.89 0.74 1 CDH11 129 1009 NM_001797 −1.56 −0.69 0.663 0.90 0.73 1 NME3130 4832 NM_002513 −1.71 0.12 0.668 1.02 0.65 1 SERPINB12 131 89777NM_080474 −1.66 0.63 0.668 1.09 0.61 1 MT3 132 4504 NM_005954 −1.77−0.16 0.672 0.97 0.69 1 PRPH2 133 5961 NM_000322 −1.69 −0.56 0.675 0.910.74 1 MCL1 134 4170 NM_021960 −1.74 −0.13 0.679 0.98 0.69 1 F7 135 2155NM_000131 −2.01 −0.06 0.679 0.99 0.69 1 C19orf20 136 91978 NM_033513−1.69 0.87 0.684 1.13 0.61 1 CASC5 137 570820 NM_020380 −1.86 −0.560.686 0.92 0.75 1 B3GNT6 138 192134 NM_138706 −1.80 0.45 0.691 1.07 0.651 SAMD3 139 154075 NM_152552 −1.99 0.68 0.692 1.11 0.62 1 HRH2 140 3274NM_022304 −1.68 −0.39 0.693 0.93 0.74 1 NDUFAB1 141 4706 NM_005003 −1.82−0.20 0.694 0.97 0.72 1 C10orf11 142 83938 NM_032024 −1.58 −0.06 0.7020.97 0.72 1 NLRP4 143 147945 NM_134444 −1.68 0.11 0.702 1.01 0.69 1GPR15 144 2838 NM_005290 −1.58 0.72 0.708 1.11 0.64 1 TANC2 145 26115NM_025185 −1.86 0.40 0.709 1.05 0.67 1 XM_371074 (SEQ ID NO: 184)HOM-TES- 146 25900 NM_080730 −1.84 −0.09 0.711 0.99 0.72 1 103 FYCO1 147794433 NM_024513 −1.67 0.86 0.714 1.13 0.63 1 COL4A3BP 148 10087NM_005713 −1.96 0.03 0.715 1.00 0.71 1 NEDD1 149 121441 NM_152905 −1.560.59 0.721 1.08 0.67 1 PCBP3 150 54039 NM_020528 −1.69 0.15 0.727 1.020.71 1 PRPF8 151 10594 NM_006445 −5.34 −2.64 0.246 0.62 0.40 1 HIPK3 15210114 NM_005734 −3.97 −2.34 0.418 0.68 0.61 1 PFKL 153 5211 NM_002626−1.85 −0.10 0.630 0.98 0.64 1 GNG3 154 2785 NM_012202 −1.86 −0.62 0.6580.90 0.73 1

TABLE 4A Triple-negative Gene Signature Gene Entrez Gene function SymbolGene ID Accession # MAD_BPLER MAD_HMLER Apoptosis CASP4 837 NM_001225−2.36 −0.92 Apoptosis MCL1 4170 NM_021960 −1.74 −0.13 Apoptosis PDCD427250 NM_014456 −2.81 −2.25 Cell Adhesion CDH11 1009 NM_001797 −1.56−0.69 Cell Adhesion COL20A1 57642 NM_020882 −3.47 −2.58 Cell AdhesionCOL4A3BP 10087 NM_005713 −1.96 0.03 Cell Adhesion PDLIM2 64236 NM_021630−2.65 −0.34 Cell Signaling DKK4 27121 NM_014420 −1.90 −0.72 CellSignaling MTA2 9219 NM_004739 −3.71 −1.07 Cell Signaling PRPH2 5961NM_000322 −1.69 −0.56 Cell Signaling RACGAP1 29127 NM_013277 −3.61 −1.46Cell Signaling RGS9BP 388531 NM_207391 −1.58 0.53 Cell Signaling TBC1D2457465 XM_370928 −1.97 −0.32 DNA binding FIZ1 84922 NM_032836 −2.97 −0.32DNA binding PHF1 5252 NM_002636 −3.21 −0.72 DNA binding ZNF324 25799NM_014347 −2.91 −1.05 DNA binding ZNF451 26036 NM_015555 −3.07 −0.77 DNAbinding ZNF490 57474 NM_020714 −4.40 −2.66 DNA binding ZNF552 79818NM_024762 −2.35 −1.30 DNA binding ZNF574 64763 NM_022752 −3.52 −1.41 DNAbinding ZNF585A 199704 NM_152655 −2.06 −0.70 DNA binding ZNF643 65243NM_023070 −3.05 −0.52 DNA repair GNAT1 2779 NM_000172 −2.03 −0.36 DNArepair GNG3 2785 NM_012202 −1.86 −0.62 DNA repair SETX 23064 NM_015046−2.40 −0.72 DNA repair TSG101 7251 NM_006292 −2.45 0.23 G1/S transitionCCNA2 890 NM_001237 −2.79 −1.39 G1/S transition LIN37 55957 NM_019104−1.92 −0.19 G1/S transition LIN9 286826 NM_173083 −2.90 −0.72 G1/Stransition POLA1 5422 NM_016937 −2.01 −0.17 G1/S transition RB1CC1 9821NM_014781 −3.21 −1.50 Gene Expression C16orf33 79622 NM_024571 −2.67−0.58 Gene Expression C6orf151 154007 NM_152551 −1.75 1.64 GeneExpression CCDC12 151903 NM_144716 −3.22 −1.34 Gene Expression DDX4211325 NM_007372 −4.00 −1.78 Gene Expression DHX8 1659 NM_004941 −4.27−1.24 Gene Expression HIPK3 10114 NM_005734 −3.97 −2.34 Gene ExpressionHNRPK 3190 NM_002140 −3.35 −2.71 Gene Expression MRPL27 51264 NM_148571−1.57 −1.17 Gene Expression MRPS9 64965 NM_182640 −2.00 −0.45 GeneExpression POLR2B 5431 NM_000938 −4.09 −1.97 Gene Expression POLR2F 5435NM_021974 −3.46 −2.98 Gene Expression POLR2G 5436 NM_002696 −3.59 −1.68Gene Expression RBM22 55696 NM_018047 −2.37 −1.53 Gene Expression RBMX27316 NM_002139 −2.43 −0.57 Gene Expression SNW1 22938 NM_012245 −5.45−1.31 Inflammation HRH2 3274 NM_022304 −1.68 −0.39 Inflammation KIR2DL43805 NM_002255 −2.02 −0.98 Inflammation NLRP4 147945 NM_134444 −1.680.11 Inflammation NOSTRIN 115677 NM_052946 −2.06 −0.69 Metabolism B3GNT6192134 NM_138706 −1.80 0.45 Metabolism DHRS13 147015 NM_144683 −3.66−1.69 Metabolism DIO1 1733 NM_000792 −1.96 −1.39 Metabolism ETHE1 23474NM_014297 −2.19 1.03 Metabolism GAPDH 2597 NM_002046 −4.30 −0.19Metabolism GBP4 115361 NM_052941 −2.09 −0.14 Metabolism ISYNA1 51477NM_016368 −1.95 −0.82 Metabolism KCNJ6 3763 NM_002240 −1.88 0.12Metabolism MGAT2 4247 NM_002408 −2.06 −0.19 Metabolism MSRB3 253827NM_198080 −2.72 −0.86 Metabolism NDUFAB1 4706 NM_005003 −1.82 −0.20Metabolism PFKL 5211 NM_002626 −1.85 −0.10 Metabolism SCD 6319 NM_005063−3.24 −1.58 Mitosis ANAPC2 29882 NM_013366 −2.80 −1.31 Mitosis ANAPC429945 NM_013367 −2.96 −0.79 Mitosis BUB1 699 NM_004336 −2.62 −0.62Mitosis C4orf15 79441 NM_024511 −2.52 0.55 Mitosis CASC5 57082 NM_020380−1.86 −0.56 Mitosis CCDC5 115106 NM_138443 −2.18 −0.02 Mitosis CEP2755142 NM_018097 −1.99 −1.76 Mitosis DLG7 9787 NM_014750 −1.93 −0.56Mitosis NDC80 10403 NM_006101 −3.24 −2.06 Mitosis NME3 4832 NM_002513−1.71 0.12 Mitosis NUF2 83540 NM_031423 −2.68 −0.90 Mitosis PPP2CA 5515NM_002715 −2.77 −0.87 Mitosis PRC1 9055 NM_003981 −3.08 −2.40 MitosisRAN 5901 NM_006325 −5.67 −2.89 Molecular CANX 821 NM_001746 −1.92 −1.38Transport Molecular CLTC 1213 NM_004859 −2.02 0.05 Transport MolecularCOPB1 1315 NM_016451 −3.13 −2.91 Transport Molecular COPE 11316NM_007263 −3.15 −1.43 Transport Molecular DDX19B 11269 NM_007242 −4.84−2.09 Transport Molecular FYCO1 79443 NM_024513 −1.67 0.86 TransportMolecular MFSD5 84975 NM_032889 −2.81 −0.38 Transport Molecular NUP20523165 XM_058073 −2.88 −1.57 Transport Molecular RFT1 91869 NM_052859−2.82 0.04 Transport Molecular SH3GL3 6457 NM_003027 −2.67 −0.55Transport Molecular SLC24A3 57419 NM_020689 −2.23 −0.75 TransportMolecular SLC47A1 55244 NM_018242 −1.62 −0.71 Transport Molecular SLC4A557835 NM_021196 −1.94 0.63 Transport Molecular TNPO1 3842 NM_002270−1.99 −0.59 Transport Proteasome NEDD1 121441 NM_152905 −1.56 0.59Degradation Proteasome NEDD8 4738 NM_006156 −1.79 0.25 DegradationProteasome PSMA1 5682 NM_002786 −5.53 −2.21 Degradation Proteasome PSMA25683 NM_002787 −4.14 −2.79 Degradation Proteasome PSMA3 5684 NM_002788−1.72 −0.54 Degradation Proteasome PSMB4 5692 NM_002796 −6.73 −2.41Degradation Proteasome PSMC1 5700 NM_002802 −1.50 −0.45 DegradationProteasome PSMC3 5702 NM_002804 −1.77 −1.02 Degradation ProteasomePSMD14 10213 NM_005805 −3.37 −2.28 Degradation Proteasome PSMD2 5708NM_002808 −4.64 −2.67 Degradation Proteasome PSMD6 9861 NM_014814 −2.05−0.79 Degradation Proteasome PSMD7 5713 NM_002811 −3.88 −2.86Degradation Proteasome RNF11 26994 NM_014372 −1.86 1.11 DegradationProteasome UBL5 59286 NM_024292 −4.14 −2.33 Degradation Proteasome USP3910713 NM_006590 −2.13 −0.04 Degradation RNA splicing C22orf29 79680NM_024627 −3.47 −2.46 RNA splicing CDC2L5 8621 NM_003718 −2.34 0.95 RNAsplicing CDC5L 988 NM_001253 −1.60 −0.87 RNA splicing INTS12 57117NM_020395 −2.22 −0.74 RNA splicing ISY1 57461 NM_020701 −4.27 −2.95 RNAsplicing LSM2 57819 NM_021177 −2.90 −0.95 RNA splicing LSM6 11157NM_007080 −2.83 −0.41 RNA splicing LSM8 51691 NM_016200 −1.76 −0.26 RNAsplicing PRPF38A 84950 NM_032864 −3.64 −1.70 RNA splicing PRPF6 24148NM_012469 −3.13 −1.23 RNA splicing PRPF8 10594 NM_006445 −5.34 −2.64 RNAsplicing SF3B14 51639 NM_016047 −2.97 −2.06 RNA splicing TUT1 64852NM_022830 −1.91 −0.07 Unassigned F7 2155 NM_000131 −2.01 −0.06 (Bloodclotting cascade/Hemostasis/ Protein metabolism) Unassigned SERPINB1289777 NM_080474 −1.66 0.63 (coagulation) Unassigned KIF26B 55083XM_371354 −1.95 −1.15 (development) Unassigned GPR15 2838 NM_005290−1.58 0.72 (GPCRs, Class A Rhodopsin- like) Unassigned PCBP3 54039NM_020528 −1.69 0.15 (RNA binding) Unassigned MT3 4504 NM_005954 −1.77−0.16 (Target of EGFR1/TGF beta/TNF-alpha signaling, metal) UnassignedDYSF 8291 NM_003494 −2.13 0.64 (Target of IL-1 singlaing) UnassignedPCTK2 5128 NM_002595 −2.29 −0.79 (Target of TGF beta signaling)Unassigned C10orf11 83938 NM_032024 −1.58 −0.06 Unassigned C12orf1060314 NM_021640 −2.11 −0.54 Unassigned C19orf20 91978 NM_033513 −1.690.87 Unassigned C19orf29 58509 XM_375557 −2.24 −0.36 Unassigned CCDC75253635 NM_174931 −3.33 −2.06 Unassigned DCDC2 51473 NM_016356 −1.61−0.02 Unassigned DNAJC11 55735 NM_018198 −1.52 0.75 Unassigned DUS3L56931 NM_020175 −2.65 −0.71 Unassigned FAM59B 150946 XM_097977 −1.850.68 Unassigned FAM86B1 85002 NM_032916 −2.54 −2.05 Unassigned FLJ1091655258 NM_018271 −2.39 −2.48 Unassigned FLJ38984 127703 NM_152374 −1.50−0.23 Unassigned HOM-TES- 25900 NM_080730 −1.84 −0.09 103 UnassignedKIAA1143 57456 NM_020696 −2.27 0.86 Unassigned KIAA1822 84439 NM_032425−2.67 −1.27 Unassigned MANEAL 149175 NM_152496 −3.02 −2.41 UnassignedMGC39900 286527 NM_194324 −2.13 −0.87 Unassigned PDE6C 5146 NM_006204−1.98 −0.33 Unassigned SAMD3 154075 NM_152552 −1.99 0.68 UnassignedSTIM2 57620 NM_020860 −2.39 −0.99 Unassigned TANC2 26115 XM_371074 −1.860.40 Unassigned TMCO4 255104 NM_181719 −1.74 −0.01 Unassigned TMED323423 NM_007364 −2.93 −1.28 Unassigned TMEM14B 81853 NM_030969 −1.99−0.56 Unassigned TMEM219 124446 XM_378175 −3.04 −2.49 Unassigned TMEM41A90407 NM_080652 −2.70 −1.83 Unassigned YTHDC1 91746 NM_133370 −2.31−0.26 Ratio Gene function Fold_BPLER Fold_HMLER BPLER/HMLER Cherry_ScoreApoptosis 0.623 0.86 0.73 1 Apoptosis 0.679 0.98 0.69 1 Apoptosis 0.4620.63 0.73 1 Cell Adhesion 0.663 0.90 0.73 1 Cell Adhesion 0.416 0.560.74 1 Cell Adhesion 0.715 1.00 0.71 1 Cell Adhesion 0.563 0.95 0.60 1Cell Signaling 0.657 0.89 0.74 1 Cell Signaling 0.545 0.79 0.69 1 CellSignaling 0.675 0.91 0.74 1 Cell Signaling 0.361 0.77 0.47 4 CellSignaling 0.707 1.10 0.64 2 Cell Signaling 0.680 0.95 0.71 2 DNA binding0.514 0.94 0.55 1 DNA binding 0.606 0.86 0.71 1 DNA binding 0.543 0.840.65 2 DNA binding 0.516 0.86 0.60 2 DNA binding 0.285 0.59 0.49 1 DNAbinding 0.595 0.80 0.75 1 DNA binding 0.425 0.79 0.54 1 DNA binding0.642 0.89 0.72 2 DNA binding 0.493 0.92 0.54 1 DNA repair 0.627 0.950.66 1 DNA repair 0.658 0.90 0.73 1 DNA repair 0.617 0.89 0.70 1 DNArepair 0.684 1.04 0.66 3 G1/S transition 0.499 0.75 0.67 2 G1/Stransition 0.571 0.97 0.59 1 G1/S transition 0.525 0.90 0.59 1 G1/Stransition 0.724 0.97 0.74 3 G1/S transition 0.466 0.74 0.63 1 GeneExpression 0.540 0.91 0.59 1 Gene Expression 0.730 1.26 0.58 2 GeneExpression 0.502 0.78 0.64 2 Gene Expression 0.425 0.71 0.60 1 GeneExpression 0.413 0.81 0.51 4 Gene Expression 0.418 0.68 0.61 1 GeneExpression 0.358 0.55 0.65 1 Gene Expression 0.587 0.81 0.73 1 GeneExpression 0.669 0.92 0.72 2 Gene Expression 0.438 0.69 0.63 4 GeneExpression 0.417 0.56 0.74 1 Gene Expression 0.507 0.74 0.69 3 GeneExpression 0.477 0.78 0.61 2 Gene Expression 0.569 0.91 0.62 1 GeneExpression 0.335 0.75 0.45 3 Inflammation 0.693 0.93 0.74 1 Inflammation0.593 0.85 0.69 1 Inflammation 0.702 1.01 0.69 1 Inflammation 0.604 0.900.67 1 Metabolism 0.691 1.07 0.65 1 Metabolism 0.350 0.75 0.47 1Metabolism 0.573 0.78 0.74 1 Metabolism 0.641 1.17 0.55 1 Metabolism0.576 0.97 0.60 2 Metabolism 0.608 0.99 0.62 1 Metabolism 0.489 0.870.57 1 Metabolism 0.654 1.04 0.63 1 Metabolism 0.586 0.97 0.60 1Metabolism 0.571 0.85 0.67 1 Metabolism 0.694 0.97 0.72 1 Metabolism0.630 0.98 0.64 1 Metabolism 0.555 0.75 0.74 2 Mitosis 0.580 0.79 0.74 1Mitosis 0.557 0.87 0.64 1 Mitosis 0.617 0.91 0.67 2 Mitosis 0.560 1.090.52 3 Mitosis 0.686 0.92 0.75 1 Mitosis 0.583 1.00 0.59 4 Mitosis 0.5220.74 0.70 3 Mitosis 0.646 0.90 0.72 1 Mitosis 0.377 0.66 0.57 3 Mitosis0.668 1.02 0.65 1 Mitosis 0.515 0.84 0.62 2 Mitosis 0.457 0.86 0.53 2Mitosis 0.409 0.60 0.68 1 Mitosis 0.266 0.57 0.47 2 Molecular 0.587 0.800.74 1 Transport Molecular 0.570 1.01 0.57 2 Transport Molecular 0.3260.54 0.61 1 Transport Molecular 0.547 0.77 0.71 2 Transport Molecular0.304 0.66 0.46 3 Transport Molecular 0.714 1.13 0.63 1 TransportMolecular 0.540 0.93 0.58 1 Transport Molecular 0.544 0.75 0.73 3Transport Molecular 0.468 1.01 0.47 1 Transport Molecular 0.544 0.900.60 1 Transport Molecular 0.637 0.88 0.72 1 Transport Molecular 0.6130.90 0.68 2 Transport Molecular 0.672 1.11 0.61 2 Transport Molecular0.630 0.90 0.70 1 Transport Proteasome 0.721 1.08 0.67 1 DegradationProteasome 0.736 1.04 0.71 1 Degradation Proteasome 0.240 0.65 0.37 4Degradation Proteasome 0.205 0.54 0.38 2 Degradation Proteasome 0.4340.90 0.48 4 Degradation Proteasome 0.147 0.52 0.28 4 DegradationProteasome 0.476 0.91 0.52 2 Degradation Proteasome 0.399 0.81 0.49 4Degradation Proteasome 0.352 0.62 0.56 3 Degradation Proteasome 0.3430.61 0.56 3 Degradation Proteasome 0.516 0.87 0.59 3 DegradationProteasome 0.256 0.53 0.49 2 Degradation Proteasome 0.671 1.17 0.57 1Degradation Proteasome 0.337 0.64 0.53 2 Degradation Proteasome 0.6950.99 0.70 2 Degradation RNA splicing 0.400 0.62 0.64 3 RNA splicing0.656 1.13 0.58 1 RNA splicing 0.654 0.87 0.75 3 RNA splicing 0.639 0.880.72 1 RNA splicing 0.306 0.54 0.56 2 RNA splicing 0.512 0.84 0.61 3 RNAsplicing 0.593 0.93 0.64 4 RNA splicing 0.700 0.96 0.73 2 RNA splicing0.403 0.69 0.58 4 RNA splicing 0.508 0.81 0.62 3 RNA splicing 0.246 0.620.40 1 RNA splicing 0.500 0.72 0.70 2 RNA splicing 0.680 0.99 0.69 2Unassigned 0.679 0.99 0.69 1 (Blood clotting cascade/Hemostasis/ Proteinmetabolism) Unassigned 0.668 1.09 0.61 1 (coagulation) Unassigned 0.5360.82 0.65 1 (development) Unassigned 0.708 1.11 0.64 1 (GPCRs, Class ARhodopsin- like) Unassigned 0.727 1.02 0.71 1 (RNA binding) Unassigned0.672 0.97 0.69 1 (Target of EGFR1/TGF beta/TNF-alpha signaling, metal)Unassigned 0.614 1.12 0.55 1 (Target of IL-1 singlaing) Unassigned 0.5520.87 0.64 1 (Target of TGF beta signaling) Unassigned 0.702 0.97 0.72 1Unassigned 0.644 0.90 0.71 1 Unassigned 0.684 1.13 0.61 1 Unassigned0.625 0.94 0.67 3 Unassigned 0.474 0.65 0.72 1 Unassigned 0.571 1.000.57 1 Unassigned 0.656 1.11 0.59 2 Unassigned 0.571 0.89 0.64 1Unassigned 0.714 1.10 0.65 1 Unassigned 0.491 0.70 0.70 2 Unassigned0.440 0.62 0.71 1 Unassigned 0.693 0.97 0.72 2 Unassigned 0.711 0.990.72 1 Unassigned 0.631 1.14 0.56 1 Unassigned 0.563 0.77 0.73 2Unassigned 0.464 0.64 0.72 1 Unassigned 0.647 0.87 0.74 1 Unassigned0.621 0.95 0.66 1 Unassigned 0.692 1.11 0.62 1 Unassigned 0.597 0.830.72 2 Unassigned 0.709 1.05 0.67 1 Unassigned 0.638 0.99 0.64 1Unassigned 0.537 0.80 0.68 2 Unassigned 0.636 0.90 0.71 1 Unassigned0.458 0.66 0.70 1 Unassigned 0.457 0.73 0.63 1 Unassigned 0.567 0.960.59 3

TABLE 4B Classification of Genes in TGS Gene Signature Cell Cell DNAG1/S Gene Apoptosis Adhesion Signaling binding DNA repair transitionExpression CASP4 CDH11 DKK4 FIZ1 GNAT1 CCNA2 C16orf33 MCL1 COL20A1 MTA2PHF1 GNG3 LIN37 C6orf151 PDCD4 COL4A3BP PRPH2 ZNF324 SETX LIN9 CCDC12PDLIM2 RACGAP1 ZNF451 TSG101 POLA1 DDX42 RGS9BP ZNF490 RB1CC1 DHX8TBC1D24 ZNF552 HIPK3 ZNF574 HNRPK ZNF585A MRPL27 ZNF643 MRPS9 POLR2BPOLR2F POLR2G RBM22 RBMX SNW1 Molecular Proteasome RNA InflammationMetabolism Mitosis Transport Degradation splicing Unassigned HRH2 B3GNT6ANAPC2 CANX NEDD1 C22orf29 F7 KIR2DL4 DHRS13 ANAPC4 CLTC NEDD8 CDC2L5SERPINB12 NLRP4 DIO1 BUB1 COPB1 PSMA1 CDC5L KIF26B NOSTRIN ETHE1 C4orf15COPE PSMA2 INTS12 GPR15 GAPDH CASC5 DDX19B PSMA3 ISY1 PCBP3 GBP4 CCDC5FYCO1 PSMB4 LSM2 MT3 ISYNA1 CEP27 MFSD5 PSMC1 LSM6 DYSF KCNJ6 DLG7NUP205 PSMC3 LSM8 PCTK2 MGAT2 NDC80 RFT1 PSMD14 PRPF38A C10orf11 MSRB3NME3 SH3GL3 PSMD2 PRPF6 C12orf10 NDUFAB1 NUF2 SLC24A3 PSMD6 PRPF8C19orf20 PFKL PPP2CA SLC47A1 PSMD7 SF3B14 C19orf29 SCD PRC1 SLC4A5 RNF11TUT1 CCDC75 RAN TNPO1 UBL5 DCDC2 USP39 DNAJC11 DUS3L FAM59B FAM86B1FLJ10916

TABLE 5 Perturbagens inducing a TGS-related signature (CMAP) PercentRank CMAP name Mean N Enrichment p-value Specificity non-null 3trichostatin A 0.381 182 0.439 0 0.4218 70 4 methylergometrine 0.604 40.896 0.0001 0 100 8 piribedil 0.581 4 0.83 0.00127 0.0089 100 11deferoxamine 0.432 8 0.593 0.00309 0 87 13 sulfametoxydiazine 0.57 40.793 0.00354 0.0094 100 15 diltiazem 0.543 5 0.716 0.00433 0 80 16nabumetone 0.526 4 0.78 0.0044 0.0065 100 18 meptazinol 0.548 4 0.7640.00585 0.0079 100 19 remoxipride 0.53 4 0.759 0.00631 0.015 100 21adrenosterone 0.48 4 0.735 0.00965 0.0217 100 24 canavanine 0.534 30.821 0.01148 0 100 25 phthalylsulfathiazole 0.504 5 0.662 0.011780.0974 80 26 butacaine 0.438 4 0.718 0.01281 0.0158 100 27 gabapentin0.482 4 0.718 0.01307 0 100 37 vorinostat 0.339 12 0.42 0.0187 0.6181 6640 sulfabenzamide 0.369 4 0.683 0.02174 0.0288 75 43 conessine 0.454 40.678 0.02335 0 75 45 adenosine 0.317 4 0.677 0.02381 0.0124 50phosphate 46 loperamide 0.288 6 0.566 0.02459 0.1616 66 49fludroxycortide 0.441 5 0.608 0.02784 0.0238 80 51 tacrine 0.339 4 0.6640.02896 0.0126 75 52 flufenamic acid 0.428 6 0.554 0.02994 0.0365 83 54practolol 0.435 4 0.657 0.03209 0.0259 75 56 4,5- 0.554 2 0.869 0.034930.0407 100 dianilinophthalimide 59 0316684-0000 0.298 4 0.644 0.038930.0192 50 60 resveratrol 0.332 9 0.439 0.04238 0.4363 55 62STOCK1N-28457 0.461 3 0.717 0.04465 0.0174 100 65 pipemidic acid 0.387 30.715 0.04543 0.0238 66 66 hesperetin 0.486 5 0.571 0.04588 0.0325 80 67vigabatrin 0.313 3 0.712 0.04649 0.1529 66 69 triamterene 0.374 5 0.5690.04724 0.0403 60 70 biotin 0.352 3 0.711 0.04745 0.0074 66 72 amikacin0.365 4 0.627 0.04828 0.0089 50 75 CP-690334-01 0.177 8 0.454 0.049240.2 50 76 methyldopa 0.391 5 0.566 0.04948 0.031 60 77 cetirizine 0.4414 0.625 0.04957 0.0857 75 78 5252917 0.545 2 0.843 0.04982 0.0959 100

TABLE 6 CMAP transcripts decreased in breast cancer cells aftertreatment with Trichostatin A (Instance ID 6709) Probe Set ID GeneSymbol Rank Score Amplitude 201267_s_at PSMC3 22265 0.001 −1.66200796_s_at MCL1 22207 −0.002 −1.46 209055_s_at CDC5L 21939 0.004 −1.19AFFX- GAPDH 21909 0 −1.17 HUMGAPDH/ M33197_5_at 215424_s_at SNW1 21890−0.005 −1.16 202731_at PDCD4 21746 −0.005 −1.07 204162_at NDC80 21731−0.01 −1.07 201705_at PSMD7 21564 −0.008 −1.01 211162_x_at SCD 21329−0.003 −0.92 207318_s_at CDK13 21150 −0.001 −0.87 215509_s_at BUB1 21032−0.001 −0.84 203418_at CCNA2 20991 −0.005 −0.83 211708_s_at SCD 20983−0.011 −0.83 208853_s_at CANX 20890 −0.012 −0.81 207319_s_at CDK13 20833−0.015 −0.79 212861_at MFSD5 20698 −0.015 −0.77 218134_s_at RBM22 20612−0.017 −0.75 203444_s_at MTA2 20562 −0.032 −0.74 206474_at CDK17 20587−0.027 −0.74 210759_s_at PSMA1 20591 −0.022 −0.74 202576_s_at DDX19A ///20554 −0.037 −0.73 DDX19B 221573_at C7orf25 /// PSMA2 20330 −0.033 −0.7209226_s_at TNPO1 20264 −0.036 −0.68 201102_s_at PFKL 20192 −0.044 −0.67203101_s_at MGAT2 20193 −0.038 −0.67 218009_s_at PRC1 20166 −0.049 −0.67221918_at CDK17 20122 −0.052 −0.66 212247_at NUP205 20070 −0.056 −0.65211565_at SH3GL3 20018 −0.059 −0.64 201532_at PSMA3 19951 −0.062 −0.63213226_at CCNA2 19947 −0.068 −0.63 AFFX- GAPDH 19837 −0.068 −0.62HUMGAPDH/ M33197_M_at 204862_s_at NME3 19691 −0.068 −0.6 213762_x_atRBMX 19570 −0.068 −0.58 217829_s_at USP39 19438 −0.068 −0.56 208852_s_atCANX 19388 −0.071 −0.55 204219_s_at PSMC1 19242 −0.07 −0.53 200749_at —18994 −0.065 −0.5 218204_s_at FYCO1 18978 −0.07 −0.5 218220_at C12orf1018967 −0.075 −0.5 215954_s_at C19orf29 18872 −0.077 −0.49 200750_s_atRAN 18834 −0.087 −0.48 221452_s_at TMEM14B 18856 −0.082 −0.48215792_s_at DNAJC11 18761 −0.089 −0.47 219119_at NAA38 18734 −0.094−0.47 210148_at HIPK3 18651 −0.096 −0.46 203334_at DHX8 18517 −0.101−0.45 211746_x_at PSMA1 18546 −0.097 −0.45 209449_at LSM2 18476 −0.105−0.44 202243_s_at PSMB4 18410 −0.108 −0.43 209511_at POLR2F 18356 −0.111−0.43 218660_at DYSF 18347 −0.116 −0.43 207657_x_at TNPO1 18249 −0.124−0.42 208837_at TMED3 18268 −0.119 −0.42 201317_s_at PSMA2 18181 −0.126−0.41 201676_x_at PSMA1 18061 −0.127 −0.4

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We claim:
 1. A method of identifying a candidate therapeutic agentagainst a cancer initiating cell comprising the step of a. exposing ahuman breast primary epithelial cell transformed derivative (BPLER) cellculture to a test agent, wherein said BPLER cell culture comprises humanbreast primary epithelial cells (BPE) transformed with at least onegenetic element selected from hTERT, SV40 early region and h-RAS^(V12);b. measuring expression of at least ten malignancy associated responsesignature biomarkers wherein the at least 10 biomarkers are selectedfrom the expression signature biomarker set consisting of SEQ IDNOs:1-154 in the BPLER cell culture to obtain a measured expressionlevel, c. comparing the measured expression level from step (b) to anexpression signature reference from a BPLER cell culture that has notbeen exposed to the test agent, wherein a decrease of expression of atleast 5 of the at least 10 biomarkers in the test culture compared tothe expression signature reference indicates that the test agent is acandidate therapeutic agent against a cancer initiating cell.
 2. Themethod of claim 1, wherein the cancer initiating cell is a basal-likebreast tumor-initiating cell.